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15.1.2: Gram Positive Cocci - Biology

15.1.2: Gram Positive Cocci - Biology


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The pathogenic Gram-positive cocci comprise two main groups of bacteria: the Staphylococci and Streptococci (the genus Enterococcus is grouped with the Streptococci). The only true pathogen in the genus Staphylococcus is Staph. aureus, but it is a nosocomial pathogen of great concern. There are multiple pathogens in the Streptococci, but only four of the most relevant Streptococcal pathogens to this course are addressed here: Strep. pyogenes, the viridans Streptococci, and Enterococcus spp. (faecalis and faecium).


MICROBIOLOGY OF ODONTOGENIC INFECTIONS Medical Assignment Help

The bacteria that cause infection are most commonly part of the indigenous bacteria that normally live on or in the host. Odontogenic infections are no exception, because the bacteria that cause odontogenic infections are part of the normal oral flora: those that comprise the bacteria of plaque, those found on the mucosal surfaces, and those ,found in the gingival sulcus. They are primarily aerobic gram-positive cocci, anaerobic gram-positive cocci, and anaerobic gram-negative rods. These bacteria cause a variety of common diseases, such as dental caries, gingivitis, and periodontitis. When these bacteria gain access to deeper underlying tissues, as through a necrotic dental pulp or through a deep periodontal pocket, they cause
odontogenic infections. Many carefully performed microbiologiC studies of
odontogenic infections have demonstrated the microbiologic composition of these infections. Several important factors must be noted. First, almost all odontogenic infections. are caused by multiple bacteria. The polymicrobial
nature of these infections makes it important that the clinician understand the variety of bacteria that are likely to

Causative Organisms”
Aerobic’
Anaerobic only’
Mixed’
Num

er I:!f Patients
28
133
243
7
33
60

In 404 patients data from Aderhold L, Konthe H, Frenkel GThe baCteriology of dentogenous pyogenic infections, OralSu:g 52:583,
1981Bartlett JG,O’Keefe P: The bacteriology of perimandibular space infections, J 6ral Surg 50: 130, 1980 ‘Chow Aw, Roser 5M, Brady FA: Orofacial odontogenic infections, Ann Intern Med 88:392, 978 lewis MAO et al: Prevalence of penicillin resistant bacteria in

e suppurative oral infection, J Antimicrob Chemothe 35B:785, 5- McCowan DA: Is antibiotic prophylaxis required far dental’ oat

.”.o’Ith joint replacement? Br Dent J 158:336, 1985 Norden

. PrNentiof’l of bone andjomt infections, Am J Med78:229, i985. cause the infection. In most odontogenic intectlons the
laboratory can identify an average of five species of bacteria. It js Dot unusual for as many as eight different species to be identified in a given infection. On rare occasions a single species may be isolated. A second important factor is the anaerobic-aerobic characteristic of the bacteria causing odontogenic infec- . < tions. Because the mouth flora is ,a combination of aerobic and anaerobic bacteria, it is not surprising to find that most odontogenic infections have both anaerobic and aerobic bacteria. Infections caused by only aerobic. bacteria probably account for 5% of all odontogenic infections. Infections caused by only anaerobic bacteria make
up about 35% of the infections. Infections caused by both anaerobic and aerobic bacteria comprise about 60% of all odontogenic infections (Table 15-1). The aerobic bacteria that cause odontogenic infections consist of many species (Table 15-2).<The most common causative organisms are streptococci, which comprise about 90% of the aerobic bacterial species that cause odontogenic infections. Staphylococci account for about
5% of the aerobic bacteria, and many miscellaneous bacteria contribute 10/0 or less. Rarely found bacteria include group D Streptococcus organisms, Neisseria spp., Corynebac

Microorganisms Causing Qdontogenic Infections”

Organism, Percentage
Aerobict 25 .
Gram-positive cocci 85
Streptococcus spp: 90
Streptococcus (group D) spp. 2
Staphylococcus spp. 6
Eikenella spp. ‘:2
Gram-negative cocci (Neisseria spp.) 2
Gram-positive rods (Corynebacterium spp.) 3
Gram-negative rods (Haemophilus spp.) . 6
Miscellaneous and undifferentiateCl 4
Ancierobict 75
Gram-positive cocci 30
Streptococcus spp. 33
Peptostreptococcus spp. 65
Gram-negative cocci (Veillonella spp.) 4
Gram-positive rods 14
Eubacterium spp.
Lactobacillus spp.
Actinomyces spp.
Clostridia spp.
Gram-negative rods 50
Bacteroides 75
Fusobacterium spp. 25
Miscellaneous 6

The anaerobic bacteria that cause infections include an even greater variety of species (see Table 15


Talk:Gram-positive bacteria

It has a lot of value actually. No article is ever complete, and this one is just less complete than some others. Mushin talk 12:33, 20 July 2006 (UTC) Correct, no article is ever done, but it still needs to be redone -- it's more a series of GP bacteria than an explanation of what it means to be GP. --Sugarskane 15:04, 20 July 2006 (UTC) Mushin is right, this is actually very important. I know I'm replying to a two-year old message. Some of you probably aren't even active. I know I had to look this up for science fair. DarkestMoonlight (talk) 14:44, 17 April 2008 (UTC)

the Gram Negative page states "Many species of Gram-negative bacteria are pathogenic," -- are gram-positive species less likely to be pathogenic? (I would consider this very useful information for someone who is not familiar with the subject). Rdchambers 08:17, 25 February 2007 (UTC)

This should now be addressed in the article. Schu1321 (talk) 23:04, 16 May 2008 (UTC)

I removed the following section from the classfication section (at least temporarily) as it seemed somewhat confusing and was unreferenced. I will look into reworking it and adding it back in, if someone else wants to look at it or revert that change, feel free. Schu1321 (talk) 22:48, 16 May 2008 (UTC)

"If the second membrane (of Gram-negative bacteria) is a derived condition, the two may have been basal among the bacteria otherwise they are probably a relatively recent monophyletic group. They have been considered as possible ancestors for the archaeans and eukaryotes, both because they are unusual in lacking the second membrane and because of various biochemical similarities such as the presence of sterols."

Actinobacteria (including Corynebacterium) are not Firmicutes: In the original bacterial phyla, the Gram-positive organisms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Staphylococcus, Streptococcus, Enterococcus, (which are cocci) and Bacillus, Corynebacterium, Nocardia, Clostridium, Actinobacteria, and Listeria (which are rods and can be remembered by the mnemonic obconical). —Preceding unsigned comment added by 160.94.159.165 (talk) 20:11, 16 June 2010 (UTC)

I believe that there should be a thorough explanation of the effects of GP vs GN bacteria on different antibiotics and the relative effectiveness of these antibiotics on the bacteria (i.e. how some are less effective on GN bacteria because they attack peptidoglycan and not the reproductive parts of the bacteria). At least a link should be posted to the antibiotic article that has this info (Actually, I'm not sure if it does have this info. ) 98.110.150.204 (talk) 00:51, 6 March 2009 (UTC)

Invite correction, to add to professionalism of the appearance of this article. Prof D. Meduban (talk) 17:12, 30 July 2011 (UTC)

Done. Stepto > Strepto. --Squidonius (talk) 22:10, 30 July 2011 (UTC)

The graphic titled "Species identification hierarchy in clinical settings" should say Streptococcus not Steptococcus. — Preceding unsigned comment added by Jason.Rafe.Miller (talk • contribs) 23:33, 22 September 2014 (UTC)

with added firmness, on the basis of 16S and other molecular phylogenetic information. Prof D Meduban (talk) 17:13, 30 July 2011 (UTC)

Not sure what you mean by "added firmness" via phylogeny. Bacterial phyla contains several cladograms that could be copied. I can envision a small cladogram of Firmicutes orders with coloured lines to indicate Gram strain (maybe using the new cladex template), but I am not sure how that would help. The section can be however expanded greatly and there is a review paper which deals with cell structure ( Sutcliffe, I. C. (2010). "A phylum level perspective on bacterial cell envelope architecture". Trends in Microbiology. 18 (10): 464–470. doi:10.1016/j.tim.2010.06.005. PMID 20637628. ) --Squidonius (talk) 23:42, 30 July 2011 (UTC)

This article contains multiple inaccuracies and out of date emphasis. "Monera" idea is long out of date, unneeded here. An S-layer is not a "membrane." It is misleading to say that most human pathogens are Gram positive, as there are plenty of Gram negative pathogens (Salmonella, V. cholerae etc.). --Joan Slonczewski, microbiologist at Kenyon College — Preceding unsigned comment added by 107.8.51.204 (talk) 15:52, 1 April 2012 (UTC)

This article, well written as it is, is way too complicated for the average Wikipedia reader. I have some medical breakdown and even with that I couldn't follow most of it. I think it needs to be thoroughly re-written so that it reads more like a patient package insert and less like a pharmaceutical textbook.

Risssa (talk) 22:05, 22 February 2014 (UTC)

Standard form used by the US Federal Government's Center for Disease Control is as follows: [1]

It would be helpful for this article, Gram-negative bacteria, and Gram staining to be standardized accordingly. Wikiuser100 (talk) 15:03, 17 March 2014 (UTC)

See similar thread at Talk:Gram-negative bacteria > Standardized spelling & punctuation. Quercus solaris (talk) 02:48, 12 June 2014 (UTC) Mix-and-matching these styles is not permissible on Wikipedia, per MOS:ARTCON. Wikipedia capitalizes eponyms and other proper names, and has no special "do not capitalize if adjectival" rule we don't care if CDC does have one. Source usage is inconsistent, so follow WP's style manual when writing at WP. — SMcCandlish ☏ ¢ > ʌ ⱷ҅ⱷ ʌ < 01:03, 20 October 2017 (UTC)

The cartoon for gram positive and negative cell wall structure is confusing. It labels the red coloured bacteria as Gram positive when this article itself says that Gram positive bacteria retain the crystal violet stain and appear purple. The structures are correct but the colouring is counter-intuitive at best.— Preceding unsigned comment added by 86.152.72.132 (talk) 11:53, 29 March 2016 (UTC)

The comment(s) below were originally left at Talk:Gram-positive bacteria/Comments , and are posted here for posterity. Following several discussions in past years, these subpages are now deprecated. The comments may be irrelevant or outdated if so, please feel free to remove this section.

I was reading this article about acid in cashews, and it said something about the acid pertaining to Gram-Positive bacteria.

Last edited at 11:54, 30 September 2010 (UTC). Substituted at 16:34, 29 April 2016 (UTC)

The following was recently added to the Pathogenesis (then renamed "Clinical Considerations"):

"Gram positive bacteria present in the lumen of the gastrointestinal tract have been found to be of benefit in the probiotic treatment of inflammatory bowel disease"

which was referenced with this review of trials investigating the effects of probiotics on various inflammatory bowel diseases. I removed the new material because it seems like this is not a conclusion of the cited paper. The only mention of gram-positive bacteria I could find in the paper was a single mention where they note that a single primary study had reported that a group given a Bifidobacterium/Lactobacillus intervention had reduced ulcerative colitis relapse over a control group. They mention a whole bunch of other studies, and conclude

"In conclusion, larger well-designed RCTs are needed to further determine whether probiotics, type of probiotics, and/or synbiotics are of clear benefit for both the induction and/or the maintenance of remission in UC."

That said, I'm certainly not opposed to having information about gram-positives as probiotics here. If someone has time to dig up some good sources, I'd be happy to help put together a section on it. Anyway, just wanted to explain the removal of material so the adder didn't think it was a knee-jerk reaction. Ajpolino (talk) 01:42, 20 May 2016 (UTC)

Please don't make assertion that lower case gram is the conventional way of writing. It is not. Just because some organization recommends it does not make it so. A quick check on Pubmed shows that the lowercase form is the less common one, probably only around a quarter of the first 100 papers I checked - [2]. It is normal to use the more common form in Wikipedia (therefore uppercase Gram-positive), for example as recommended in WP:COMMONNAME. Hzh (talk) 23:43, 19 October 2016 (UTC)

  • "Gram" is someone's name, isn't it? The convention on WP and just about everywhere else is to cap the item. Tony(talk) 23:01, 18 October 2017 (UTC)

As noted in earlier thread: Mix-and-matching these styles ("Gram stain", "gram-negative") is not permissible on Wikipedia, per MOS:ARTCON. Wikipedia capitalizes eponyms and other proper names, and has no special "do not capitalize if adjectival" rule we don't care if CDC does have one. Source usage is inconsistent, so follow WP's style manual when writing at WP. — SMcCandlish ☏ ¢ > ʌ ⱷ҅ⱷ ʌ < 01:04, 20 October 2017 (UTC)

HiSMcCandlish this usage has already been dealt with - see Orthographic note on both pages. --Iztwoz (talk) 16:57, 20 October 2017 (UTC)

The orthographic note is about inconsistent source usage off of WP it has nothing to do with how WP itself is written, which is to its own style manual. — SMcCandlish ☏ ¢ > ʌ ⱷ҅ⱷ ʌ < 22:28, 20 October 2017 (UTC)

The capitalization matter, which people have apparently been editwarring about since 2004, is being addressed at an RfC (also "advertised" at WP:VPPOL):

The result of the above RfC was "no consensus". Elsewhere, SMcCandlish wrote: "The scientific literature, like the general book-publishing market, strongly prefer Gram-negative over Gram negative, gram-negative, or 'gram negative. There is no way around this. You can blame me for this if you like (my skin is thick), but it won't change the facts." This appears to be accurate. Also see MOS:EPONYM and Wikipedia talk:Manual of Style#MOS:EPONYM See straw poll below for the current local consensus. --Guy Macon (talk) 13:31, 1 December 2020 (UTC)

Per MOS:EPONYM, Should capitalized eponyms (named after Hans Christian Gram) lose their capitalization when used adjectivally? --Guy Macon (talk) 13:31, 1 December 2020 (UTC)


Clinical Value

Global Burden of Disease

The burden of infectious diseases globally is enormous with approximately a third of deaths globally due to communicable diseases. Although tremendous progress has been made during this past century to reduce morbidity and mortality due to infectious diseases, especially in developed countries, the percentage of deaths due to infectious diseases compared to many other types of human disease is still quite significant. Diseases that have challenged us for centuries, such as tuberculosis and cholera, still infect and kill many people each year. Although there has been a worldwide campaign to eradicate diseases such as polio, this disease remains to be eliminated from the causes of human disease. While long-standing causes of infectious diseases remain prevalent, new emerging infectious diseases add to the challenge of physicians treating patients. Agents including West Nile virus, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome coronavirus, H5N1 avian influenza virus, HIV, and human metapneumovirus have all appeared or spread widely in recent times. The threat of a pandemic, in which the disease occurs over a very large geographic area, is real, although not a guarantee, and vigilance by public health practitioners and infectious disease specialists around the world will aid in curtailing the next emerging disease. Beyond the confirmed associations of infectious agents with many diseases, there are many cases for which there are limited data to associate a specific disease with an infectious process, and thus, much ongoing research attempts to further determine these associations. More than thirty diseases have some evidence suggesting an infectious agent may be involved, but either no agent has been identified or the evidence is still preliminary. Examples include Crohn's disease, psoriasis, sarcoidosis, diabetes mellitus, chronic fatigue syndrome, and atherosclerosis.

Association of Viruses with Cancer

Surprising to many is the finding that microbes play a role in several malignancies that were previously thought to be unrelated to infectious diseases. Helicobacter pylori has been shown to have a likely role in gastric cancer, and human papillomavirus is a cause of cervical and head and neck cancers. Human herpes virus 8 is a causative agent of Kaposi's sarcoma and Epstein�rr virus causes lymphoma and possibly Hodgkin's disease. It seems likely, given this change in our understanding of the relationship between cancer and microbes, that additional examples will be discovered.

Role of the Microbiology Laboratory in Accurate Diagnosis

An infectious disease can present with signs and symptoms consistent with multiple noninfectious disease processes, so a positive microbiology result can be the key information to guide therapy. There are multiple dramatic cases of patients who have undergone surgery for treatment of a noninfectious disease or who were being evaluated for malignancies when, unexpectedly, the patients had treatable infectious processes. Effective communication between primary treating physicians, the infectious disease consult team, and microbiology laboratory staff helps to ensure that correct tests are being ordered, with additional stains and media added as needed to cover the differential diagnosis. Furthermore, the correct interpretation of data is required to decide optimal therapy, with test limitations conveyed from laboratory staff to the treatment team. Some typical examples of disease associations with specific bacteria are listed in Table 2 .

Table 2

Examples of bacteria𠄽isease associations

OrganismDisease
ActinomycesCervicofacial, thoracic, abdominal, central nervous system infections
Burkholderia cepacia complexPulmonary infections
CampylobacterGastroenteritis
Clostridium perfringensFood poisoning, myonecrosis, soft tissue infections
EikenellaHuman bite wounds
EnterococcusUrinary tract infections, bacteremia
Enterohemorrhagic E. coliWatery diarrhea, hemolytic uremic syndrome
Haemophilus influenzaeMeningitis, septicemia, cellulitis, respiratory infections, otitis media
Helicobacter pyloriGastritis, duodenal ulcers, gastric adenocarcinoma
Klebsiella pneumoniaePneumonia, urinary tract infections
Legionella pneumophilaPneumonia
Listeria monocytogenesMeningitis, bacteremia
Mycoplasma pneumoniaeAtypical pneumonia
Neisseria gonorrhoeaeGonorrhea, septic arthritis, pelvic inflammatory disease
Pseudomonas aeruginosaPulmonary, skin/soft tissue, burns, bacteremia
SalmonellaDiarrhea, enteric fever
Staphylococcus aureusSuppurative skin infections, bacteremia, osteomyelitis, pneumonia, toxin-mediated infections
Streptococcus pyogenesSuppurative infections, pharyngitis, scarlet fever, skin and soft tissue infections, toxic shock-like syndrome, rheumatic fever, glomerulonephritis

Outbreaks

Outbreaks involve many people each year. In 2009�, the US public health departments reported 1527 foodborne disease outbreaks, resulting in 29򠑄 cases of illness, 1184 hospitalizations, and 23 deaths. The steps taken to manage a new outbreak include the detection of an outbreak strain in a patient by the clinical microbiology laboratory, followed by investigation of the source of the infection, and virulence, susceptibility, and genetic analysis in public health and research laboratories.

Nosocomial Infections

Nosocomial infections are those acquired in the healthcare system. They are very common and are often associated with the most resistant organisms, making therapy difficult. The infection control team in a healthcare facility monitors nosocomial infections and adherence to isolation guidelines, along with investigation of potential outbreaks or organism transmission within an institution. The diagnostic microbiology laboratory performs testing for surveillance and for transmission investigation. Urinary tract infections are the most common nosocomial infection, followed by surgical wound and respiratory tract infections, and then finally bloodstream infections. New hospital monitoring systems such as the National Healthcare Safety Network now require hospitals to participate in tracking of nosocomial infections. Tracking patients who are colonized with specific resistant organisms, following by implementation of isolation precautions while hospitalized, is of enormous value in protecting susceptible and vulnerable patients from additional infections that could be spread throughout the hospital by healthcare workers. Examples include methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, vancomycin-resistant Enterococcus, respiratory viruses, and multidrug-resistant Gram-negative enterobacteriaceae such as Klebsiella pneumoniae that are positive for the carbapenemase enzyme, blaKPC.

Legislative Considerations

Some infections have become a major concern for public health agencies and to the general public. Given the spread of MRSA in recent years, some state governments in the United States have established legislation requiring specific types of monitoring in hospitals, such as in the intensive care units. Implementation of this legislation was very controversial, as many physicians felt that although important to monitor, action would be best decided by a hospital's infection control team based on data in that hospital, instead of being legislated by the regional government without the flexibility of adjusting quickly to changing needs for monitoring infections as trends change. Flexibility would permit assigning resources to the most essential areas of concern at any one time. Many countries have guidelines established by consensus committees of infectious disease organizations to control the spread of resistant organisms, even if there is no specific legislation regarding this issue.

Susceptibility Testing

Susceptibility testing is one of the most critical functions of the microbiology laboratory. It is best to use the minimal number of drugs that will effectively treat an infection and physicians often start broad-spectrum drugs as empirical therapy until further laboratory data are available. If the broad-spectrum antimicrobial agent is not replaced with the optimal drugs, toxicity may be worse, and spread of resistance throughout the world is a very significant consequence of widespread use of antibiotics.

Multiple drugs to treat bacteria, fungi, viruses, and parasites have been developed since the 1940s however, microbes have evolved to counter the effectiveness of these agents in many cases. Resistance has developed and spread due to mutations in genomes or plasmids, small circular DNA strands that replicate independently of the chromosome, combined with the transfer of resistance genes from one species to another. Some strains of resistant bacteria are very common, such as methicillin-resistant S. aureus, vancomycin-resistant Enterococcus, and penicillin-resistant S. pneumoniae. Some multidrug-resistant organisms are resistant to all available antimicrobial agents in our arsenal, which is devastating for the patients who get these infections.

By identifying an infectious agent as the cause of a set of symptoms, a patient may be spared further unnecessary invasive procedures to diagnose his condition. Clearly, the accurate diagnosis of the responsible infectious agent is of extreme importance to properly treat individual patients, to follow and stop the spread of disease within the population, and to increase our scientific understanding of the pathogenesis of both old and new infectious diseases. Further, staying up-to-date on improvements in our diagnostic assays and advances in technology is a key part of the job of a medical microbiologist.


15.1.2: Gram Positive Cocci - Biology

Christopher Mary Anthony1, Nyoyoko Veronica Fabian2*, Isong Blessing Asuquo1

1Akwa Ibom State University, Nigeria

2Department of Microbiology, University of Nigeria, Nigeria

Corresponding author: Nyoyoko Veronica Fabian, Department of Microbiology, University of Nigeria, Odili Hall, Nsukka, Nigeria. Tel: +08136421103 E-mail: [email protected]

Received Date: 18 September, 2020 Accepted Date: 28 September, 2020 Published Date: 05 October, 2020

Infections due to staphylococci are of major importance to human medicine. S. aureusis one of the most significant pathogens causing diseases world­wide. S. aureusis the leading cause of nosocomial infections and is responsible for a wide range of human diseases, including endocarditis, food poisoning, toxic shock syndrome, septicemia, skin infections, soft tissue infections and bone infections, as well as bovine and bovine mastitis. This study was designed for the characterization of S. aureus from urine samples of Urinary Tract Infected (UTI) students from Akwa Ibom State University. The result of this study indicates that of the 100 urine samples collected from both male and female students, 73 samples of urine were positive for S. aureus which accounted for 73% of samples collected. Sex distribution of S. aureus in this study showed a higher prevalence among the female students 40 (40%) while the male students had a prevalence of 33 (33%). High resistance was recorded in Norfloxacin, Chloramphenicol and Streptomycin with mean zone of inhibition 24 mm each. On the other hand, high sensitivity was observed in Levofloxacin, Gentamycin and Erythromycin each with average zone of inhibition of 25, 23 and 22 mm respectively. This is followed by Ampiclox and Rifampicin with average zones of inhibition of 21 and 20 mm respectively.

Introduction

Urinary Tract Infections (UTI), is an infection caused by the presence and growth of microorganism in the urinary tract. This infection is associated with bacteruria and pyuria [1]. Some of the risk factors of UTI include gender, sexual activity, immune system disorder, urinary tract anatomical malformations, disruption of normal flora of the genital area with antiseptics and antibiotics, urinary catheter and instrumentation [2-4]. About 95% of urinary tract infection occurs when bacteria ascend the urethra to the bladder or ascend the ureter to the Kidney. Urinary tract infection occurs much more frequently in female than male due to the proximity of the urethra to the anus. Approximately 50% of all women will have at least one UTI in her life time with many women having several infections through their life time [2].

Gram negative bacteria have been found most frequently in UTI cases by several authors with E. coli and Klebsiella pneumonia being the most predominant organisms [5]. Other bacteria pathogens frequently isolated include S. aureus, S. epidermidis and Streptococcus faecalis [6]. The degree and prevalence of occurrence of one or two of these organisms over others depends on the environment [7]. UTI may be classified into urethritis, cystitis and pyelonephritis depending on the site of infection. The commonest mode of infection is the ascending route, through which organisms of the bowels flora contaminates the urethra, ascends to the bladder and migrates to the kidney or prostrate.

Treatment regimen of UTI has been based largely on antibiotics and occasional surgery which is done to drain abscesses, correct underlying structural abnormalities or relieve obstructions. This study was aimed at evaluating the frequency of the most causative agents of UTI and the antimicrobial susceptibility pattern of the pathogenic isolates of the urinary tract. A number of different microorganisms have been known to cause urinary tract infections these include those of the normal flora of the skin, genital areas, anus and those from exogenous sources that maybe contacted through bad sanitary habits especially of underwear&rsquos [3]. Some of the risk factors of UTI include gender, sexual activity, immune system disorder, urinary tract anatomical malformations, disruption of normal flora of the genital area with antiseptics and antibiotics, urinary catheter and instrumentation [2,3]. About 95% of urinary tract infection occurs when bacteria ascend the urethra to the bladder or ascend the ureter to the Kidney. Urinary tract infection occurs much more frequently in female than male due to the proximity of the urethra to the anus. Approximately 50% of all women will have at least one UTI in her life time with many women having several infections through their life time [2]. Members of the genus staphylococcus are Gram positive cocci that tend to be arranged in grapelike cluster [8]. Staphylococcus aureus is a Gram-positive spherical bacterium approximately 1 &mum in diameter. Its cells form grape-like clusters, since cell division takes place in more than one plane. It is often found as a commensal associated with skin, skin glands, and mucous membranes, particularly in the nose of healthy individuals. It has been estimated that approx. 20-30% of the general population are S. aureus carriers [9]. On a rich medium, S. aureus forms medium size &ldquogolden&rdquo colonies. On sheep blood agar plates, colonies of S. aureus often cause &beta-haemolysis [8]. The golden pigmentation of S. aureus colonies is caused by the presence of carotenoids and has been reported to be a virulence factor protecting the pathogen against oxidants produced by the immune system [9].

Staphylococci are facultative anaerobes capable of generating energy by aerobic respiration, and by fermentation which yields mainly lactic acid. Staphylococcus sp. is catalase-positive, a feature differentiating them from Streptococcus sp., and they are oxidase-negative and require complex nutrients, e.g., many amino acids and vitamins B, for growth. S. aureus is very tolerant of high concentrations of sodium chloride, up to 1.7molar. S aureus may be pathogenic or nonpathogenic and the pathogenic strains are usually coagulase-positive and cause disease in their hosts. The infection may manifest as abscesses or mastitis to a severe toxic shock syndrome [9]. Microorganisms are ubiquitous in nature, and have been known resides in the human body either on the skin, in the gut or other locations. Several parts of the body serves as microflora for different microorganisms, but when found in an area different from their microflora, they could cause harm. Microorganisms such as Staphylococcus aureus which has the skin or genital area as its microflora are likely to migrate to area such as the urinary tract most especially if proper hygiene is not taken or could be transmitted through fomites such as toilets facilities. This study is set to examine urinary tract infection cases among students caused by Staphylococcus aureus and to determine the antibiotics that could help eliminate it.

Urinary tract infection

Urinary Tract Infections (UTI) caused by the presence and growth of microorganism in the urinary tract is associated with bacteriuria and pyuria. Bacteriuria is defined as the presence of bacteria in the urine while pyuria is defined as the presence of pus in the urine [1]. A Urinary tract infection can occur anywhere along the Urinary tract. It is classified as urethritis, cystitis, pyelonephritis [10]. Urethritis is the Infection of urethra with bacteria, protozoa, viruses, or fungi. This occurs when organisms gain an access to it periurethral glands in the bulbous and pendulous portions of the male urethra and in the entire female urethra. Many sexually transmitted pathogens like Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonasvaginalis, and herpes simplex virus are common causes in both sexes [11]. While Cystitis is the infection of the bladder. It is more common in women, in which cases of uncomplicated cystitis are usually preceded by sexual intercourse. It is also defined as significant bacteriuria with associated bladder mucosal invasion, and is distinguished from asymptomatic bacteriuria by the presence of symptoms such as dysuria, urgency, frequency, nocturia, haematuria and suprapubic discomfort in a febrile woman with no evidence of systemic illness [11], and Pyelonephritis is a condition suggested when at least 100,000 bacteria /ml of a single uropathogen in a midstream MSSU culture is identified with associated inflammation of the renal parenchyma, calices and pelvis in the presence of systemic illness. It can progress to maternal sepsis, preterm labour and premature delivery. Symptoms include flank or renal angle pain, pyrexia, rigor, chills, nausea, vomiting and hip pain. Symptoms of lower tract infection, such as frequency and lack of urination, may or may not be present [12].

Route of Bacteria Invasion in the Urinary Tract

There are two important routes by which bacteria can invade and spread within the urinary tract. These pathways are: ascending and haematogenous pathways. There is little evidence to support a lymphatic spread of infection to the urinary tract with any regularity (Sobiezcyk, 2011). Haematogenous Route is a route of Infection in which the renal parenchyma is being infected by blood-borne organisms which occur in humans, the haematogenous route is less common than the ascending route. For patients that suffers bacteraemia or endocarditis, the kidney is the site in which abscess occur caused by Staphylococcus aureus which is a Gram positive organism infections of the kidney with Gram negative bacilli rarely occur by the haematogenous route (Sobiezcyk,2011). Ascending Route, most uropathogens originate in the rectal flora and enter the bladder via the urethra. The female urethra is short and proximal to the vulvar and perineal areas, making contamination likely. In women in whom urinary tract infection develop, the urethra is colonized and the uropathogen gains entry to the bladder, presumably by means of the urethral massage that accompanies sexual intercourse. Whether infection develops depends upon the particular organism, the size of the inoculums, and the adequacy of host defences. Once the bacteria ascend into the bladder they may multiply and then pass up the ureters, particularly if vesicoureteral reflux is present, to the renal parenchyma (Sobieszczyk, 2011).

Signs and Symptoms of Urinary Tract Infection

Urethritis is the main symptoms are dysuria and urethral discharge. Discharge can be purulent, whitish, or mucoid. Characteristics of the discharge, such as the amount of purulence, do not reliably differentiate gonococcal from non-gonococcal urethritis [13]. Cystitis is another symptoms and the onset is usually sudden, typically with frequency, urgency, and burning or painful voiding of small volumes of urine. Nocturia, with suprapubic pain and often low back pain, is common. The urine is often turbid, and microscopic hematuria can occur. A low-grade fever may develop. Pneumaturia can occur when infection results from avesicoenteric or vesicovaginal fistula or from emphysematous cystitis. Since the frequent urge to urinate is common during pregnancy, it may be hard to tell the presence of cystitis, especially if symptoms are mild. A doubt of an infection should be clarified, because untreated cystitis puts the patient at high risk for getting a kidney infection, especially while pregnancy [13]. In acute pyelonephritis, symptoms may be the same as those of cystitis. One third of patients have frequency and dysuria. However, with pyelonephritis, symptoms typically include chills, fever, flank pain, colicky abdominal pain, nausea, and vomiting. If abdominal rigidity is absent or slight, a tender, enlarged kidney is sometimes palpable. Costovertebral angle percussion tenderness is generally present on the infected side [13].

Staphylococcus aureus

Staphylococcus aureus has been implicated as the most notorious organism associated with nosocomial infection. It is a Gram positive versatile aerobe which is a normal flora of the skin but when opportune cause many infections of which nosocomial infection have posed a scourge to pregnant women over the years [14]. S. aureus is a Gram positive coccus that occurs in grape-like clusters. It is a eubacterium found on the surface of the human skin and mucous membranes [15]. It is also found in other areas of human contact such as air, dust and food products [15]. describes Staphylococcus species can be described as a group of non-motile, non-capsulate, Gram positive cocci of uniform size (about 1&mum in diameter) that occur characteristically in clusters/groups but also singly and in pairs and also an opportunistic pathogen in man and animals [14]. Most strains become infectious usually when the skin or mucous membrane is punctured by variety of objects such as needles, blades, surgical devices etc. Little wonder then, that it is a serious threat within the hospital. Staphylococcus aureus raises greater concern because it is a pathogen with high virulence [16], high ability to cause a diverse array of life threatening infections, and its capacity to adapt to different environmental conditions [17].

Pathogenesis of Staphylococcus aureus

  1. aureus is both a commensal organism and a pathogen. The anterior nares are the main ecological niche for S. aureus. Approximately 20% of individuals are persistently nasally colonized with S. aureus, and 30% are intermittently colonized. However, numerous other sites may be colonized, including the axillae, groin, and gastrointestinal tract. Colonization provides a reservoir from which bacteria can be introduced when host defences are breached, whether by shaving, aspiration, insertion of an indwelling catheter, or surgery. Colonization clearly increases the risk for subsequent infection [18]. In a study of bacteraemia, blood isolates were identical to nasal isolates in 82% of patients. Colonization also allows S. aureus to be transmitted among individuals in both healthcare and community settings. The basis for S. aureus colonization is complex and incompletely understood but appears to involve the host&rsquos contact with S. aureus (e.g., other carriers) and the ability of S. aureus to adhere to host cells and to evade the immune response [18]. In establishing an infection, S. aureus has numerous surface proteins, called &ldquoMicrobial Surface Components Recognizing Adhesive Matrix Molecules&rdquo (MSCRAMMs) that mediate adherence to host tissues. MSCRAMMs bind molecules such as collagen, fibronectin, and fibrinogen, and different MSCRAMMs may adhere to the same host-tissue component. MSCRAMMs appear to play a key role in initiation of endovascular infections, bone and joint infections, and prosthetic-device infections. Different S. aureus strains may have different constellations of MSCRAMMs and so may be predisposed to causing certain kinds of infections [19]. Once S. aureus adheres to host tissues or prosthetic materials, it is able to grow and persist in various ways. S. aureus can form biofilms (slime) on host and prosthetic surfaces, enabling it to persist by evading host defences and antimicrobials [20]. The ability to form and reside in biofilms is one reason why prosthetic- device infections, for example, can be so difficult to eradicate without removal of the device. In vitro, S. aureus can also invade and survive inside epithelial cells, including endothelial cells, which theoretically may also allow it to escape host defences, particularly in endocarditis [21]. S. aureus is also able to form Small-Colony Variants (SCVs), which may contribute to persistent and recurrent infection. In vitro, SCVs are able to &ldquohide&rdquo in host cells without causing significant host-cell damage and are relatively protected from antibiotics and host defences. They can later revert to the more virulent wild-type phenotype, possibly resulting in recurrent infection [22]. Its main defences are production of an ant phagocytic microcapsule (most clinical isolates produce type 5 or 8). The zwitterionic capsule (both positively and negatively charged) can also induce abscess formation. The MSCRAMM protein A binds the Fc portion of immunoglobulin and, as a result, may prevent opsonisation. S. aureus may also secrete chemotaxis inhibitory protein of staphylococci or the extra- cellular adherence protein, which interfere with neutrophil ex- travasation and chemotaxis to the site of infection [23]. In addition, S. aureus produces leukocidins that cause leukocyte destruction by the formation of pores in the cell membrane [24]. During infection, S. aureus produces numerous enzymes, such as proteases, lipases, and elastases that enable it to invade and destroy host tissues and metastasize to other sites. S. aureus is also capable of producing septic shock. It does this by interacting with and activating the host immune system and coagulation pathways. Peptidoglycan, lipoteichoic acid, and a-toxin may all play a role [21]. In addition to causing septic shock, some S. aureus strains produce super antigens, resulting in various toxinoses, such as food poisoning and toxic shock syndrome [25]. Regulation of expression of staphylococcal virulence factors plays a central role in pathogenesis. To reduce undue metabolic demands, expression occurs in a coordinated fashion only when required by the bacterium. Expression of MSCRAMMs generally occurs during logarithmic growth (replication), whereas secreted proteins, such as toxins, are produced during the stationary phase. During infection, the early expression of the MSCRAMM proteins facilitates initial colonization of tissue sites, whereas the later elaboration of toxins facilitates spread. The Accessory Gene Regulator (AGR) is a quorum-sensing system that plays a critical role in the regulation of staphylococcal virulence [26]. The AGR mutants appear to have diminished virulence, and certain AGR types are associated with particular clinical syndromes (Cheung, Eberhardt and Chung, 1994). Among those who developed S. aureus bacteremia and non-carriers had mortality higher than that among carriers. Because most infections among carriers occurred with their colonizing strains, colonization may confer some protective immunity if staphylococcal infection develops [27]. As described, S. aureus has numerous mechanisms to produce disease and to evade host defences. However, it is important to note that not all S. aureus strains are created equal. Different strains may contain different adhesins or toxins or may differ in their ability to produce biofilms and resist phagocytosis. The distribution of some virulence factors is related to clonal type, whereas the presence of others is unrelated to genetic background [28]. In this regard, it is important to note that there is limited information on the expression of these genes during infection.

Innate Host Defence against S. aureus Infections

Polmorpho nuclear leucocytes (PMNs) are arguably the most important cellular defences against invading bacteria, such as S. aureus. Host innate defences fight bacteria by using two mechanisms namely: killing of bactreia and phagocytosis of the bacteria. Killing of bacteria, PMN phagocytosis is followed by the execution of bactericidal mechanisms, including the production of superoxide radicals and other Reactive Oxygen Species (ROS), and enrichment of antimicrobial peptides, proteins, and degradative enzymes in the phagosome. ROS are generated by a multicomponent membrane-bound complex known as the NADPH-dependent oxidase, which is defective in individuals with chronic granulomatous disease [29]. In resting neutrophils, components of the NADPH oxidase are either cytosolic (p40 phox , p47 phox , p67 phox , and the GTPase Rac2) or located in membranes (flavo-cytochrome b558). NADPH oxidase assembly involves translocation of the cytosolic protein components to the plasma or phagosome membrane and their subsequent association with flavocytochrome b558, a transmembrane heterodimer that serves as the nidus of the assembling enzyme complex. After activation of the NADPH oxidase, electrons are transported from cytosolic NADPH to molecular oxygen, thereby generating superoxide anion [29]. Multiple oxygen metabolites, including hydrogen peroxide, superoxide anion, and hypochlorous acid, contribute to neutrophil bactericidal activity [30]. Phagocytosis of bacteria neutrophils are recruited rapidly to the site of infection and remove invading microorganisms through a process known as phagocytosis. Bacteria express a litany of molecules on their surface, such as lipopolysaccharide, lipoprotein, and lipoteichoic acid, and these pathogen-associated molecular patterns interact with receptors on the surface of neutrophils. In general, ligation of the neutrophil pattern recognition receptors (e.g., Toll-like receptors and CD14) activates signal transduction pathways that ultimately contribute to bactericidal activity. PMN phagocytosis is most efficiently promoted by opsonisation of bacteria with antibody and complement. Specific anti-body binds to epitopes on the surface of bacteria and enables the deposition of complement. Antibodies bound to the bacterial surface are recognized by neutrophil receptors. In addition to activation of PMN oxygen-dependent bactericidal mechanisms, phagocytosis triggers degranulation, which involves fusion of cytoplasmic granules with the phagosome membrane. Peroxidase-negative granules, including secretory vesicles, gelatinase granules, and specific granules, are a reservoir of functionally important membrane proteins, such as CR3, formyl peptide receptor, flavocytochrome b558, and b2-integrins [31]. Peroxidase-positive granules (primary/azurophilic granules) contain the bulk of oxygen-independent antimicrobial agents of neutrophils, including a-defensins, cathepsins, proteinase-3, elastase, azurocidin, lysozyme, and bacteri-cidalpermeabilityincreasing protein [31].

Staphylococcus aureusas a Perpetuator of Urinary Tract Infection during Pregnancy

Staphylococcus aureus is an opportunistic pathogen affecting both immune competent and immunocompromised individuals, frequently resulting in high morbidity and with complications, which constitute problem to health care institutions. This bacterium has been reported by several studies as the causative organism of wide varieties of diseases of supportive infections such as boil, wound infection, pustule and urinary tract infections [32]. It is also the most common cause of infection in hospitals with high prevalence among new born babies, surgical patients, malnourished persons and patient with diabetics [33]. This bacterium is known to be notorious in the acquisition of resistance to new drugs and continues to defy attempt at medical control and the prevalence of multi drug methicillin resistant S. aureus with very limited treatment choice is also on the increase. Many strains of S. aureus carry wide variety of multi drug resistant genes on plasmids which aid the spread of resistance even among different species [34]. In most urinary tract infections cases in Nigeria, symptomatic patients usually indulged in discriminate usage of antibiotics before consulting the physicians when they no longer control the symptomatic situations [35]. Physicians on the other hand treat the patients with wide broad spectrum antibiotics without any microbiological investigations [36]. This widespread indiscriminate use and inappropriate prescription of antibiotics in the treatment of urinary tract infections are significant contributing factors to the emergence and spread of bacterial resistance to the commonly used antimicrobial agents [37]. This situation is worsening with the high prevalence of fake and substandard antimicrobial agent in Nigeria markets [38].

Materials and Methods

Study Population

The study population was drawn from Akwa Ibom State University Students, Akwa Ibom State, Nigeria. Urine samples were collected from total of 100 students (50 male and 50 female).

Samples Collection

A total of 100 clean urine samples were collected in sterile universal containers from 50 male and 50 female students of all age groups respectively with suspected cases of UTI. At the point of collection, samples were labeled appropriately and immediately taken to the microbiology laboratory and investigated for microscopy, culture and sensitivity analysis.

The urine from each samples were inoculated into plates of Mannitol Salt agar using sterile swab stick. All plates were incubated at37°C aerobically for 24 hrs. The plates examined macroscopically for bacterial growth.

Isolation of Staphylococcus aureus

The presumptive colonies of S. aureus on Mannitol salt agar plates were further cultured on Nutrient agar plates and repeatedly sub-cultured to get pure culture. These isolates recovered were preserved for further bacterial identification.

Bacterial Identification

The isolates were identified as S. aureus on thebasis of Gram staining, colony morphology on Mannitol Salt Agar (MSA) and haemolysis patterns on Blood Agar. Biochemical tests include Catalase, Oxidase, Coagulase, Indole and Citrate tests which were done on the basis of conventional biochemical test with reference [39,40], while Gram staining was done according to the method described [39].

Gram staining: Slides with heat fixed smear were placed on a staining rag. The smears was gently flooded with crystal violet and were left to stand for 1 minute the slides were tilted slightly and gently rinsed with tap water using wash bottle. The smears were again gently flooded with Gram&rsquos iodine and were left to stand for 1 minute the slides were tilted slightly and gently rinsed with tap water using a wash bottle. The slides were tilted slightly and alcohol was flooded for 5 seconds and was immediately rinsed with water. The smears were flooded gently with safranin to counter- stain and were left to stand for 45 seconds. The slides were tilted and gently rinsed with tap water using a wash bottle and was blot dry with bibulous paper and allowed to air dry. The stained slides with smears were viewed using light microscope under oil immersion.

Biochemical test: All Gram positive cocci were subjected to the following biochemical test: Catalase test and Coagulase test.

Catalase test: A small amount of bacterial colony was transferred to a surface of the clean glass slide and dry slide using an inoculating loop. A drop of hydrogen peroxide was placed on the slide and mixed with the colony, A positive result was a rapid evolution of oxygen gas (within 5-10 seconds) as evidenced by bubbling. A negative result lacked bubbles or few bubbles scattered.

Coagulase test: Drops of normal saline were placed on a grease free slide. Colonies of a 24h old culture was emulsified on the drop of normal saline, a drop of plasma was added and mixed gently. Clumping within 10 seconds indicated positive coagulase test while absence of clumping indicated negative coagulase test.

Antimicrobial Sensitivity Test

The antibacterial susceptibility testing was done using Kirby-Bauer NICCS modified disc agar diffusion technique: all procedure was done under aseptic technique. Isolated bacteria were subculture in normal saline for 3 hours interval to obtain a solution with turbidity equal to 0.5 McFarland standards. Sterile swab stick was used to evenly spread the organism across the Muller Hinton agar plate and allowed to dry. The impregnated disks were carefully picked with sterile forceps and carefully placed on the inoculated Muller Hinton agar plate. The plates were incubated at 37 0 c for 24 hours. The diameters of the zones of inhibition were measured and recorded for the urine samples that showed zones of inhibition. The zones of inhibition were measured using a meter rule. All measurements were recorded in millimeters. The Sensitivity pattern of the isolates to Levofloxacin (20 mcg), Erythromycin (30 mcg), Streptomycin (30 mcg), Ampiclox (20 mcg), Gentamicin (10 mcg), Rifampicin (20 mcg), Amoxillin (20 mcg), Norfloxacin (10 mcg), Ciprofloxacin (10 mcg) and Chloramphenicol (30 mcg), were determined. Isolates were divided into three groups based on the zone of inhibition produced by the antibiotic disc susceptible, intermediately susceptible and resistant according to the Clinical and Laboratory Standards Institute (CLSI) guideline Performance Standards for Antimicrobial Susceptibility Testing (2007).

In this study, a total of 100 urine samples were aseptically collected from 50 male and 50 female students in Akwa Ibom State University.

Table 1 presents the distribution of S. aureus from the samples collected. The result shows that S. aureus was isolated from 33 (33%) out of the 50 samples collected for men and 40 (40%) out of 50 samples collected from female patients. This give a total of 73 (73%) samples of which S. aureus was isolated from out of the 100 samples collected.

For the samples of which S. aureus were isolated, culture identification, Gram&rsquos staining and biochemical tests were carried out. Table 2 shows the result of Gram&rsquos staining biochemical tests such catalase, coagulase, oxidase, hemolysis, citrate and indole. The result revealed that the pure cultures exhibited clusters of Gram positive cocci. All the isolates were positive for catalase and coagulase test while negative for Oxidase and indole test. They were also positive for hemolysis.

Table 3 shows the sensitivity pattern of the isolate against some antibiotics, the result shows that in the present investigation, most samples were resistant to Norfloxacin, sample M13 was resistant to all the antibiotics used and sample M16 was sensitive to all the antibiotics.

Infections due to staphylococci are of major importance to human medicine. S. aureus is one of the most significant pathogens causing diseases worldwide [41]. S. aureus is the leading cause of nosocomial infections and is responsible for a wide range of human diseases, including endocarditis, food poisoning, toxic shock syndrome, septicemia, skin infections, soft tissue infections and bone infections, as well as bovine and bovine mastitis.

This study was designed for the characterization of S. aureus from urine samples of Urinary Tract Infected (UTI) students from Akwa Ibom State University.

The result of this study indicates that of the 100 urine samples collected from both male and female students, 73 samples of urine were positive for S. aureus which accounted for 73% of samples collected. Table 1 shows that the sex distribution of S. aureus in this study showed a higher prevalence among the female students 40 (40%) while the male students had a prevalence of 33 (33%) which support previous reports [42,43]. This observation could be due to the anatomical nature of the women urethra and its proximity to the anus which favor the fecal and skin flora easy access to the urethra. However, the relationship between men and women usually favors the transfer of this organism leading to the increasing level of prevalence among the men.

Identification of Staphylococcus aureus was based on cultural characteristics, Gram staining and biochemical properties. All the 73 isolates fermented Mannitol with the color change of Mannitol Salt Agar (MSA) and produced yellow colony, they also showed &beta-haemolysis on blood agar media enriched with 5% human blood (Table 2). Gram stained smears of the pure cultures exhibited clusters of Gram-positive cocci. These isolates were positive for catalase and coagulase test while negative for Oxidase test (Table 2). In catalase test Hydrogen peroxide was broken down into water and oxygen. Production of oxygen was indicated by bubble formation. The isolates were identified as S. aureus by coagulase test. The positive result of coagulase test was confirmed by the formation of curd like clotting compared to negative control. The isolates were also identified as Oxidase negative, the result was confirmed due to its inability to give blue color on reaction with oxidase reagent. Earlier findings [44-46], identified and characterized Staphylococcus aureus on the basis of cultural characteristics, Gram staining and Biochemical characterization.

This study showed the presence of Staphylococcus aureus as one of the possible etiologic agents of Urinary tract infection cases. This finding was inconformity with that of Amengialue et al. [44] who reported Staphylococcus aureus as the most prevalent bacteria among UTI patients which accounted for 73% of all isolates. Similar reports were earlier reported [42, 47-50]

In this study, high resistance was recorded in Norfloxacin, Chloramphenicol and Streptomycin with mean zone of inhibition 24 mm each. On the other hand, high sensitivity was observed in Levofloxacin, Gentamycin and Erythromycin each with average zone of inhibition of 25, 23 and 22 mm respectively. This is followed by Ampiclox and Rifampicin with average zones of inhibition of 21 and 20 mm respectively. These findings are slightly correlated to that of Yabaya et al. [45], they observed that Staphylococcus aureus is sensitive to Ciprofloxacin, Streptomycin and Erythromycin. On other hand this result is contrary to that of Jahan et al. [46], who revealed that Staphylococcus aureus is resistant to Erythromycin.

Urine samples from different Urinary tract infected students in Akwa Ibom State University, Nigeria were tested. Isolates were confirmed as S. aureus by Cultural characteristic, Gram staining and Biochemical tests. The isolates were resistant to Norfloxacin, Chloramphenicol and Streptomycin. On the other hand, the isolates were found to be sensitive against Levofloxacin, Gentamycin and Erythromycin. This study showed that S. aureus is the most frequent etiologic agent of urinary tract infection. Resistance pattern against broad spectrum antibiotic depicts an alarming situation, which needs special attention. High susceptibility S. aureus isolates to gentamicin and ciprofloxacin was observed in this study, this is an indication that these antibiotics can be used for empirical treatment of infections from orthopedic patients in this institution since vancomycin is not readily available in this community. The S. aureus isolates were highly resistant to beta lactam antibiotics. The abuse of beta-lactam antibiotics and other classes of antibiotics in our community should therefore be controlled.

This study will be useful for the identification of the bacteria and its sensitivity pattern to some antibiotics, which will provide vital information on how to prevent the disease and as well to improve public health system in Akwa Ibom State University and Nigeria at large.

It is recommended that antibiotic susceptibility test should be conducted to Urinary tract infection patient before prescription in order to avoid antibiotic resistance.

Table 1: Distribution of Staphylococcus aureus recovered.


Figure 1

Figure 1. Overall structure of BshC from B. subtilis. (A) Schematic of the BshC domains. The N-terminal portion of the Rossmann fold (NTR) is colored green. The CP1 and CP2 domains are colored light blue and purple, respectively. The C-terminal portion of the Rossmann fold (CTR) is colored green. The α-helical coiled-coil domain is colored red. (B) Stereo representation of the BshC monomer with the domains colored as they are in panel A. The ligands are shown as spheres, with citrate situated within the canonical active site in front of the Rossmann fold parallel β-sheet and ADP bound between the CP1 and coiled-coil domains. All macromolecular graphics were prepared with PyMOL.(12)

The BshC crystals used in this study were grown in the presence of 200 mM citrate, and electron density corresponding to a citrate molecule was observed within the canonical Rossmann fold active site (Figure S2 of the Supporting Information). The 5- and 6-carboxylate groups of citrate likely mimic the 4- and 1-carboxylate groups, respectively, of the malyl moiety of glucosaminyl-malate and BSH. Thus, this citrate molecule provides clues about how BshC accommodates that portion of its substrate and product (Figure 3A). The 5- and 6-carboxylate groups of citrate interact with the guanidinium groups of Arg 377 and Arg 504 and main chain amide groups of Gly 352 and Glu 353, respectively. The remaining citrate carboxylate group is less ordered and makes no direct interactions with the enzyme. It extends into a solvent-filled cavity, which has enough space to accommodate larger ligands. Electron density corresponding to a glycerol molecule was observed near the citrate (Figure S2 of the Supporting Information). This glycerol molecule interacts with the side chain of Glu 353 and an ordered water molecule (Figure 3A).


Clinical Situations of Bacteriology and Prognosis in Patients with Urosepsis

Background. Urosepsis and septic shock are a critical situation leading to a mortality rate up to 30% in patients with obstructive diseases of the urinary tract. Aim. To analyze the bacterial distribution and drug resistance of pathogenic bacteria in patients with urosepsis and to provide a basis for the rational application of antibacterial drugs in clinical practice. Methods. A retrospective analysis of 94 hospitalized patients with urosepsis for 6 years was performed. The strain composition, resistance characteristics, and the antibiogram of common bacteria from positive blood and midstream urine culture were analyzed. Results. A total of 87 strains were isolated, including 65 strains (74.71%) of Gram-negative bacilli, 14 strains (16.09%) of Gram-positive cocci, and 8 strains (9.20%) of fungi. The Gram-negative bacilli included 42 strains of Escherichia coli (E. coli) (64.62%), among which 34 strains (80.95%) were producing ESBLs, and 14 strains (21.84%) of Klebsiella pneumoniae (K. pneumoniae), among which nine strains (64.29%) were producing ESBLs. The most common pathogenic bacteria, ESBL+ E. coli and K. pneumoniae strains, showed sensitivity towards imipenem, ertapenem, piperacillin/tazobactam, amikacin, and cefotetan, but were highly resistant to quinolones. The cure rate of urosepsis was 88.30%, and the susceptibility rate of septic shock was 45.47%. Significance. Gram-negative bacterial infections are the main cause of urosepsis. The mild patient group showed more E. coli (ESBL-) infections, and the number of ESBL producing E. coli isolated from the mild group showed higher drug resistance rates for aztreonam and levofloxacin compared with isolates from the severe group.

1. Introduction

Sepsis is a global public health problem, and it is also one of the most common critical infectious diseases, with a mortality rate as high as 20-42%. Approximately 215,000 patients in the United States die of septic shock every year, among which 9.1% were infected with an etiological source from the urogenital system [1]. Urosepsis is a life-threatening organ dysfunction resulting from systemic metabolic imbalance in response to the infection, which normally originates from the urogenital tract of the host [2].

Urinary system diseases, including urinary tract obstruction and associated iatrogenic surgical injury, may often predispose the patients to develop secondary infections of varying etiology [3]. Due to the complexity of urinary tract obstruction, secondary infections may occur in the presence of urethral stones or hydronephrosis that lead to the formation of bacterial biofilms. Second, many invasive surgical procedures such as local puncture of the urinary system can cause serious damage to the normal skin and mucous membrane barriers [4]. Most of the current surgical methods are based on intraluminal invasive procedures and, for example, percutaneous nephrolithotomy (PCNL) involving high pressure irrigation and exosmose of the irrigating fluid can lead to the destruction of the tissue structure [5]. Other invasive procedures such as transrectal prostate biopsy can lead to damage of the intestinal mucosal barrier, and the intestinal flora entering the blood can increase the risk of sepsis and subsequent septic shock. Once the urinary tract infection progresses into urinary septic shock, the mortality rate is greatly increased [6].

A number of studies have been conducted to explore the epidemiological characteristics of urinary tract infections and sepsis, but there is still a lack of relevant bacteriological characteristics and prognostic analysis of urosepsis in China [7]. Some global research reports suggested that the most common pathogenic bacteria isolated from nosocomial urosepsis caused by urinary tract infections were mostly Escherichia coli, Enterococci, Pseudomonas aeruginosa, and Klebsiella spp., which are highly resistant to the routinely used antibiotics (among these pathogenic microorganisms, 8% were resistant to imipenem and 62% to beta-lactamase inhibitors) [8]. Although the European guidelines for urinary tract infections provided valuable guidance on the diagnosis and treatment of various urinary tract infections, there are still great differences in the pathogenic spectrum and susceptibility results of urosepsis around the world [9].

Clinically, in terms of the principles for antibacterial treatment in urosepsis patients with unknown etiology, stratified diagnosis of the risk factors for drug-resistant bacteria should be performed based on the epidemiological data of local monitoring and assessment of pathogens and its drug resistance and to initiate and perform timely empirical treatment [10]. This study retrospectively analyzed the distribution and drug resistance characteristics of common pathogenic bacteria isolated from blood and urine of patients with urosepsis in a hospital in China. The outcomes and prognosis of infected patients were analyzed. We hoped to predict the relationship between the clinical severity of urosepsis and the pathogenic bacteria and their drug resistance, in order to help the clinicians improve their understanding and clinical prognosis of patients with urosepsis. Moreover, our analysis may provide theoretical basis and relevant data for the rational use of antibiotics and guide the clinical treatment of urinary tract infections.

2. Patients and Methods

2.1. Patients

A retrospective study of data collected from the patients with urosepsis who were admitted to the Department of Urology and ICU, Fujian Provincial Hospital, from January 2012 to December 2017, was conducted. The study protocol was approved by the hospital ethics committee.

2.1.1. Inclusion Criteria

Urinary tract infections were diagnosed according to the Health Industry Standards of the People’s Republic of China WS/T 489-2016 “Laboratory Diagnosis of Clinical Microbiology of Urinary Tract Infections”: the number of pathogenic bacteria in culture of clean catch midstream urine or catheterized urine specimen was ≥10 5 CFU/L. Urosepsis was diagnosed according to the 2017 European Guidelines for Urinary Tract Infection: life-threatening organ dysfunction caused by imbalanced response to the infection in the host induced by urinary tract infections. Urosepsis can be divided into three stages: systemic inflammatory response syndrome (SIRS), sepsis, and septic shock.

2.1.2. Exclusion Criteria

(1) Patients with comorbid infection in other sites, such as pulmonary infection, catheter-related bloodstream infections (CRBSI, referred to bloodstream infection and bacteremia caused by the bacteria and fungi that colonized an intravascular indwelling catheter), abdominal infection or intracranial infections, etc. or (2) hospitalized patients due to other infections.

2.2. Diagnostic Criteria and Grouping

According to the severity of the disease, the included patients were divided into two groups: mild and severe. Urosepsis patients with early manifestation of systemic inflammatory response syndrome (SIRS) were classified as the mild group and those who progressed to sepsis or septic shock was classified as the severe group.

Systemic inflammatory response syndrome (SIRS) is a systemic nonspecific inflammatory response caused by severe damage due to infectious or noninfectious factors such as infection, trauma, burns, surgery, ischemia-reperfusion, etc., eventually leading to a cluster of clinical symptoms of uncontrolled inflammatory response in the body. Systemic reactions caused by severe infections include changes in body temperature, respiration, heart rate, and white blood cell count. The characteristics include body temperature >38°C or <36°C, heart rate >90 beats/min, respiratory rate >20 beats/min or partial pressure of arterial carbon dioxide <32 mmHg, peripheral white blood cell count >12,000/mm 3 or <4000/mm 3 .

Sepsis was defined as a life-threatening organ dysfunction caused by imbalanced response to the infection in the host, which can be indicated by the sequential organ failure assessment (SOFA) score in the clinical setting (SOFA

2 points) [11]. The quick sequential organ failure assessment (qSOFA) score is composed of three items: changes in conscious state, systolic blood pressure ≤100 mmHg, and respiratory frequency ≥22 times/min. Patients who meet two or more of these criteria items, i.e., qSOFA score ≥2) were categorized as suspected sepsis cases.

The clinical diagnostic criteria for septic shock recommended in the guidelines are persistence of hypotension after full capacity resuscitation in patients with sepsis administration of vasoconstrictive drugs to maintain the mean arterial pressure (MAP) ≥65 mmHg and serum lactate levels >2 mmol/L.

2.3. Sample Collection

Urine and blood samples from all subjects were collected, separated, and cultured in accordance with the Chinese Health Industry Standards. During the study period, blood culture, identification of bacteria, and drug sensitivity analysis were performed with the use of the BacT/Alert 3D fully automatic blood culture system (BioMerieux, France), which supports aerobe and anaerobe culture flasks, and of a VITEK 2 MS mass spectrometer, which is a fully automatic bacteria identification system. The system also allows for drug sensitivity identification, which was performed using Columbia blood agar, Maconkey agar, and MH agar (Beiruite Biotechnology (Zhengzhou) LLC, Zhengzhou, China) and drug sensitive paper (Oxoid Co., UK). Inoculation, isolation, and culture were conducted for all specimens in accordance with the National Clinical Laboratory Procedures (3rd edition). The judgment criteria were based on the Clinical and Laboratory Standards Institute (CLSI). Quality-control bacterial strains included Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, Staphylococcus aureus ATCC25923, and Staphylococcus aureus ATCC25913.

2.4. Survey Methods

A retrospective survey approach was used. The inpatient medical records were reviewed and data were collected for patients who met the criteria including age, gender, length of hospital stay, primary diseases in urinary system, underlying diseases, comorbidities, pathogenic examinations, bacterial culture, and hospital mortality.

2.5. Statistical Analysis

The data presented as means ± standard deviation, medians and range, and frequencies and percentage, as appropriate. Continuous data were analyzed using the

- test or Mann-Whitney U test, as appropriate. Categorical data were analyzed using the chi-square test. Two-sided P values <0.05 were considered as statistically significant. Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 16.0 (IBM, Armonk, NY, USA)

3. Results

A total of 94 eligible patients who met the inclusion criteria were included 47 males and 47 females, aged 17-88 years with an average of 59.83 years. Among them, 83 cases were cured, 10 cases were initiatively discharged, and one case died (Figure 1).

The 94 patients were classified into the mild group (38 cases) and severe group (56 cases). Among the mild cases, the male/female ratio was 24/14 with an average age of 58.09 years. Among severe cases, the male/female ratio was 23/33, and the average age was 62.39 years (Table 1). Of the 94 patients with urosepsis secondary to infection of urinary system, 30 cases were hospitalized in the ICU (seven mild cases and 23 severe cases). The occurrence rate of urinary sepsis shock was 45.47% (Table 1). Among the patients with urosepsis, 52 cases (57.45%) were secondary to the urinary tract obstruction with stones or hydronephrosis 38 cases (40.43%) were due to urinary surgery.

3.1. Etiologic Distribution

The blood and urine samples were collected and a total of 87 different bacterial strains belonging to 22 species were identified. Among them, there were 65 strains (74.71%) of eight species of Gram-negative bacteria 14 strains (14.09%) of nine species of Gram-positive bacteria, and eight strains (9.2%) of five species of fungi. The most common Gram-negative bacteria isolated from patients with urosepsis were E. coli (ESBLs+/-), K. pneumoniae (ELBSs-/+), Enterobacter cloacae, Stenotrophomonas maltophilia, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Acinetobacter junii. The most common Gram-positive bacteria isolated from patients with urosepsis were Enterococcus faecium, Enterococcus faecalis, Staphylococcus epidermidis, Staphylococcus capitis, human Staphylococcus subgroup, Staphylococcus saprophyticus, Aerococcus viridians, and Staphylococcus warneri. The fungi included Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, and Trichosporon asahii (Tables 2–4). The severe group showed more ESBL nonproducing Escherichia coli (E. coli ESBL-) isolates compared with the mild group (P<0.05).

3.2. Gram-Negative Bacteria

The detection rates of extended-spectrum β-lactamases (ESBLs) in E. coli and K. pneumoniae strains were 80.95% and 64.29%, respectively. The resistance rates of the main pathogenic bacteria in this group, ESBLs-producing E. coli strain (E. coli+), were all higher than 80% to most antibiotics such as ampicillin (penicillin), cefazolin (first generation cephalosporin), ceftriaxone (third-generation cephalosporin), ciprofloxacin and levofloxacin (quinolones). The drug resistance rates of this strain were 50% to aztreonam (monocyclic amides) and cefepime (fourth-generation cephalosporins). The drug resistance rates of ESBLs-producing K. pneumonia strain (K. pneumonia +) were also higher than 90% to antibiotics such as ampicillin-sulbactam, cefazolin, ceftriaxone, gentamicin, and trimethoprim/sulfamethoxazole. The drug resistance rates of the two strains were higher than 50% to gentamicin and tobramycin (aminoglycosides). Their sensitivity to carbapenems, such as imipenem and ertapenem were 100%, and they were relatively sensitive to enzyme inhibitor compound drugs such as piperacillin/tazobactam.

3.3. Gram-Positive Bacteria

The Gram-positive bacteria were mainly Enterococcus faecium and Staphylococcus. The drug resistance patterns of both Enterococcus faecium and Staphylococcus in the mild and severe groups were compared, but did not show any significant difference among them (Supplement Table 1). The drug resistance rates of Enterococcus faecium (accounting for 66.66% of the Gram-positive strains) were greater than 50% to most of the antibiotics. It was 100% resistant to antibiotics such as ampicillin, penicillin G, ciprofloxacin, levofloxacin, and moxifloxacin. One case of drug resistance to vancomycin was sensitive to linezolid and tigecycline (Table 4).

Drug resistance of Escherichia coli strains isolated from the mild and severe groups of patients were analyzed. The number of ESBL producing (ESBL+) E. coli, showing drug resistance to aztreonam (100% versus 68.75%) and Levofloxacin (100% versus 75%) isolated from the mild group was higher than in the severe groups (P<0.05). Regarding the ESBL+/- Escherichia coli isolated in the two groups, no other significant difference in antibiotic resistance to any other antibiotics was observed (Table 5). This may be due to the small sample size in this retrospective study.

4. Discussion

Urinary tract infections (UTIs) are the second most common infectious diseases after respiratory infections. Urosepsis is a life-threatening organ dysfunction caused by imbalanced response to the infections in the host due to urinary tract originated infections. It can be manifested as three stages: early SIRS, sepsis, and septic shock [9]. Patients with urosepsis can progress from almost innocuous state to severe sepsis in a very short period of time. Therefore, patients with urosepsis, especially those with complex urinary tract infections, should be diagnosed, intervened, and treated at an early stage. SIRS is commonly recognized as the first event in a cascade leading to multiple organ failure, with a significant increase in mortality [12, 13]. Treatment strategy for urosepsis should include appropriate life-sustaining and prompt antimicrobial therapy, supplementary measures, and optimal management of urinary system disorders [14]. Urosepsis is more common in men than in women, with a higher rate of detection in elderly patients, and suffering with Gram-negative bacteria as the main pathogen. The local factors include urinary tract obstruction with urethral stones comorbid with hydronephrosis, high pressure washing with percutaneous nephroscopy, and transrectal biopsy [15].

According to the monitoring results in our hospital, the pathogenic bacteria were still dominated by Gram-negative bacteria. For the strains isolated from this retrospective study, E. coli was the main pathogenic bacteria (accounting for 61.67% of all pathogenic bacteria) among Gram-negative bacteria, which was close to the results from Nicolie et al. (42-69.3%) [16].

This study suggested that the detection rates of extended-spectrum β-lactamases (ESBLs) in E. coli and K. pneumoniae strains were 80.95% and 64.29%, respectively, which were higher than in previous reports. Indeed, the 2015 CHITE test data suggested that the detection rates of ESBLs in E. coli and K. pneumoniae were 51.5% and 27.4%, respectively [17]. The production of extended-spectrum enzymes ESBLs is one of the major resistance mechanisms of E. coli and K. pneumoniae. ESBLs are a class of plasmid-mediated β-lactamases that are able of hydrolyzing penicillins, oxyiminocephalosporins (including third- and fourth-generation cephalosporins), and monocyclic amides aztreonam, and can be inhibited by β-lactamase inhibitors [18]. Early studies on the resistance mechanism of ESBLs indicated that it is formed by the point mutation of 1-7 amino acids in the TEM-1 or SHV-1 molecular structure. In recent years, the genotype of ESBLs has greatly changed. The CTX-M type had replaced the TEM and SHV as the main genotypes of ESBLs, accounting for more than 70% of all ESBLs genotypes [19]. ESBL producing strains often simultaneously carry the drug resistance genes for aminoglycosides, tetracyclines, and quinolones, which allow the spread of drug resistance genes among bacteria by binding, transformation, and transduction, resulting in occurrence of severe intrahospital cross-infection and spread of resistant bacteria outside the hospital.

E. coli and K. pneumoniae are the most common ESBLs-producing bacteria in the Enterobacteriaceae family, followed by Proteus bacteria [20]. The main pathogenic bacteria in this group, ESBLs-producing E. coli strains, had drug resistance rates of >80% to most antibiotics such as ampicillin (penicillins), cefazolin (first generation cephalosporins), ceftriaxone (third-generation cephalosporins), ciprofloxacin, and levofloxacin (quinolones). The drug resistance rates were 75% to aztreonam (monocyclic amides) and cefepime (fourth-generation cephalosporins). The resistance rates of ESBLs-producing K. pneumoniae strain were more than 80% to antibiotics such as ampicillin-sulbactam, cefazolin, ceftriaxone, gentamicin, and trimethoprim/sulfamethoxazole (Supplement Table 2). The resistance rates of these two strains were greater than 50% to gentamycin and tobramycin (aminoglycoside).

Some previously published studies [21–23] showed that the main risk factors for ESBLs-producing bacterial infections included repeated use of antimicrobial drugs, indwelling catheters (including central vein or arterial catheters, percutaneous gastric or jejunostomy fistulas), the presence of urethral stones or obstructions (biliary/urinary tract), previous ESBLs-producing bacterial infections, repeated hospitalizations (including care centers), previous admission in ICUs, elderly patients, underlying diseases (diabetes, immune dysfunction), and ventilator-assisted breathing. Meanwhile, studies have shown that the increase of mortality in patients with bloodstream infection due to ESBLs-producing enteric bacterial infection was not associated with the production of ESBLs, but was due to the inappropriate empiric treatment in patients with community-acquired bloodstream infections caused by ESBLs strains, suggesting the importance of appropriate empirical treatment for patients with sepsis [20].

In this study, the drug resistance rate of urosepsis pathogenic spectrum to quinolones was as high as 90%. Therefore, for patients with high risk factors for urosepsis (such as urinary tract stones combined with obstruction, operation of endoluminal lithotripsy, etc.), the quinolone group of drugs such as levofloxacin should be carefully selected as the first choice for the empirical administration of drugs in the early phase of treatment. Further clinical data are required for clarifying whether a combination drug therapy for ESBLs-producing strains can be recommended if in vitro susceptibility tests show the sensitivity to relevant bacterial isolate. At the same time, the drug resistance rates of ESBLs-producing strains to third- and fourth-generation cephalosporins are as high as 75% [24]. In order to reduce the multiplication and spread of ESBLs-producing strains and β-lactam antibiotic resistance, the use of third-generation cephalosporins should be restricted to perioperative prophylactics for patients with low risk for ESBLs-producing strains, and they should be avoided in empirical anti-infection regimens. Carbapenem antibacterial drugs (imipenem and ertapenem) had shown high antibacterial activity against ESBLs-producing E. coli and K. pneumoniae strains, and are currently the most effective and reliable antibacterial drugs in the treatment of infections caused by ESBLs-producing Enterobacteriaceae group [25]. However, with the increasing use of this class of drugs, even though it showed a high stabilizing effect on β-lactamase and strong antibacterial activity, it had been reported that the corresponding drug-resistant strains had emerged [25]. Meanwhile, it was easy to cause dysbacteriosis and secondary fungal infection due to the indiscriminate use of broad spectrum antibacterial drugs [26].

In this study, the drug resistance rate of ESBLs-producing strains to β-lactam inhibitors was less than 10%. Therefore, in order to avoid the abusive use of carbapenems in clinical practice, the carbapenems (imipenem, ertapenem, and meropenem) are only recommended in patients with high risk of extended-spectrum β-lactamase (ESBL) strains. For some patients, the β-lactam inhibitors may also be selected. In this study, when the extent of infection with different pathogenic bacteria was compared between the two groups, the mild group showed more ESBL nonproducing E. coli compared with the severe groups. A significantly high drug resistance against aztreonam (100% versus 68.75%) and levofloxacin (100% versus 75%) were shown by ESBL producing (ESBL+) E. coli strains isolated from the mild group compared with the strains from the severe group, which is consistent with a previous study [27].

The major limitation of the study is the small sample size, which could preclude an exact interpretation of specific patterns of drug resistance in the mild and severe group of patients. This could be solved by conducting a multicenter study involving a large number of patients.

5. Conclusion

Gram-negative bacterial infections are the most common cause of urosepsis in our study. When the mild and severe groups were compared for extent of infections with different bacterial pathogens, the mild group showed more E. coli (ESBL-) infections compared with the severe groups. Regarding the drug resistance pattern, the number of ESBL producing (ESBL+) E. coli isolated from the mild group showed higher drug resistance rates for aztreonam and levofloxacin, compared with isolates from the severe group. Treatment strategy for urosepsis should include appropriate life-sustaining and prompt antimicrobial therapy, supplementary measures, and optimal management of urinary system disorders. Quinolones should be selected as the first choice for the empirical administration of drugs in the early phase of treatment. Carbapenem antibacterial drugs should be the first-line treatment against ESBLs-producing E. coli and K. pneumoniae strains. In order to reduce the multiplication and spread of ESBLs-producing strains and β-lactam antibiotic resistance, the use of third-generation cephalosporins should be restricted to perioperative prophylactics for patients with low risk for ESBLs-producing strains, and they should be avoided in empirical anti-infection regimens.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval

The study protocol was approved by the hospital ethics committee.

Conflicts of Interest

All authors declare that they have no any conflicts of interest.

Acknowledgments

This study was supported by the National Key Clinical Specialty Construction Project Funding (2011) 170 and the Fujian Province Intensive Medical Center Construction Project (2017-510).

Supplementary Materials

Supplement Table 1: the drug resistance of Gram-positive bacteria in mild and severe groups. Supplement Table 2: the drug resistance rates of K. pneumoniae in the mild and severe groups. (Supplementary Materials)

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Copyright

Copyright © 2019 Ying Jiang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The microbiology of necrotizing soft tissue infections

Objective: A large number of necrotizing soft tissue infections (NSTI) treated at a single institution over an 8-year period were analyzed with respect to microbial pathogens recovered, treatment administered, and outcome. Based on this analysis, optimal empiric antibiotic coverage is proposed.

Methods: A retrospective chart review of all patients with documented NSTI was conducted. Microbiologic variables were tested for impact on outcome using Fisher’s exact test and multivariate analysis by logistic regression.

Results: Review of the charts of 198 patients with documented NSTI revealed 182 patients with sufficient microbiologic information for analysis. These 182 patients grew an average of 4.4 microbes from original wound cultures, although a single pathogen was responsible in 28 patients. Eighty-five patients had combined aerobic and anaerobic growth, the most common organisms being, in order, Bacteroides species, aerobic streptococci, staphylococci, enterococci, Escherichia coli, and other gram-negative rods. Clostridial growth was common but did not affect mortality unless associated with pure clostridial myonecrosis. Mortality was affected by the presence of bacteremia, delayed or inadequate surgery, and degree of organ system dysfunction on admission.

Conclusions: NSTI are frequently polymicrobial and initial antibiotic coverage with a broad-spectrum regimen is warranted. The initial regimen should include agents effective against aerobic gram-positive cocci, gram-negative rods, and a variety of anaerobes. The most common organisms not covered by initial therapy were enterococci. All wounds should be cultured at initial debridement, as changes in antibiotic coverage are frequent once isolates are recovered.


Summary

Introduction

Management of osteoarticular infections combines surgical treatment with antibiotic therapy. For some teams the immediate postoperative regimen requires at least partly wide-spectrum probabilistic treatment while waiting for the microbiological results. This protocol exposes the patient to the selection of resistant bacteria and the hospital unit to a modification of its bacterial ecology. The objective of this study was to retrospectively describe the microbial epidemiology of the Traumatology and Orthopaedics Department of the Lille University Hospital over 10 years (2002–2011).

Materials and methods

The bacterial species isolated in culture of osteoarticular samples were listed, after removing any duplicates. The antibiotics retained for follow-up were those used in treatment of these infections as well as those recognized as markers of resistance. For Gram-positive species, the antibiotics considered were methicillin, rifampicin, fluoroquinolones, glycopeptides, and linezolid for the Gram-negative species, cefotaxime, cefepime, imipenem, and fluoroquinolones were considered.

Results

Of the 5006 strains isolated between 2002 and 2011, Gram-positive cocci accounted for more than 71% Staphylococcus aureus 27%, and coagulase-negative staphylococci (CoNS) 54%. Contrary to S. aureus, resistance to methicillin, fluoroquinolones, and teicoplanin significantly increased in CoNS, reaching 44%, 34%, and 22%, respectively, of the strains in 2011. The proportion of streptococcal and enterococcal infections remained stable, a mean 7.4% and 5.3%, respectively, per year. Enterobacteria (12.5% of the isolates) were producers of extended-spectrum beta-lactamase in 7.8% of the cases. Pseudomonas aeruginosa was involved in 3.6% of the infections, and 12% of the strains remained resistant to ceftazidime. Propionibacterium acnes accounted for 5.8% of the bacteria isolated and showed few antibiotic resistance problems.

Discussion

Stability in the distribution and the susceptibility of different bacterial species was noted over this 10-year period. Although the evolution of S. aureus resistance was favourable, the resistance of CoNS specially to methicillin and glycopeptides increased.


Discussion

This study is one of the few to focus specifically on ICU patients admitted for HAIs with positive staphylococci peritoneal cultures. Only minor differences in clinical presentation were observed on admission compared to other patients. Adequacy of anti-infective therapy was less frequently achieved in patients with staphylococci-positive cultures. Unexpectedly, the S-HAI group had decreased morbidity (less persistent sepsis, fewer reoperation rate, and fewer surgical complications). However, no difference in hospital stays or survival was observed either between S-HAI and nS-HAI or between Sa-HAI and CoNS-HAI.

In our cohort, the prevalence of staphylococci in postoperative intra-abdominal infections was high compared to that observed in other reports, in which 9 to 12% of staphylococci-positive peritoneal samples were usually reported 5 , 23 , 24 . The demographic and clinical characteristics and initial severity were similar in patients with and without positive staphylococci peritoneal cultures, except that diabetes was more frequently observed in the Sa-HAI group, which is a well-established risk factor for S. aureus infections 25 . However, this link has been rarely reported in patients with intra-abdominal infections.

In our S-HAI cohort, lower proportions of enterococci, especially Enterococcus faecium, were reported. This observation suggests some kind of competition between Gram-positive cocci, which is an issue that has not been previously reported. Interestingly, no obvious link was reported between staphylococci and candida, while interactions with Candida albicans are well known for their ability to form persistent biofilms in the host or on medical devices and can lead to increased morbidity and mortality 26 . However, this is consistent with the fact that the development of biofilm in intraperitoneal infections has never been proven, except for peritonitis due to dialysis catheter infection. This comment is also valid for staphylococci and nonfermenting Gram-negative microorganisms, since collaborations between S. aureus and Pseudomonas aeruginosa have also been reported 27 . Lower proportions of Enterobacterales, Escherichia coli and Streptococcus spp. in the CoNS-HAIs compared to the Sa-HAIs may be explained by the fact that there was more ongoing antibiotics at the time of reoperation for PIs in this group.

Adequacy of EAT was decreased in the staphylococci group, mainly because of high proportions of methicillin-resistant staphylococci. These methicillin-resistant microorganisms required more combination therapy and more frequently included vancomycin as the documented antibiotic therapy. This difference was not found in the CoNS-HAIs vs Sa-HAIs comparison. Indeed, even if there were more methicillin-resistant staphylococci in the CoNS group, more than a quarter of them were not targeted by the definitive antibiotic therapy. There was more escalation of antibiotic therapy in the S-HAI and CoNS-HAI groups and less de-escalation in the CoNS-HAI group. The issue of de-escalation and escalation of antibiotics is debatable in the absence of standard definitions, resulting in variable interpretations between prescribers 28 . However, our criteria included both the spectrum of molecules used and their number. This approach makes it possible to consider the effects on both Gram-negative and Gram-positive bacteria.

The ICU mortality rate of patients with positive staphylococci cultures was similar to that observed with other microorganisms. Interestingly, morbidity criteria, such as persistent sepsis and surgical complications, seem to be less frequently reported in staphylococci patients contrary to what was formerly reported in a study focusing on methicillin-resistant S. aureus during HAIs 8 . A possible explanation for these results could be the competition within the peritoneal space between staphylococci and enterococci, and these organisms are thought to induce increased rates of morbidity and complications 29 – 31 . Thus, we can hypothesize that decreased morbidity rates could be linked to lower proportions of enterococci in the S-HAI group.

Unexpectedly, we did not find any difference in terms of prognosis between the CoNS-HAI and Sa-HAI subgroups. This result could be explained by a lack of power due to the small number of patients in these subgroups. Since all S. aureus were treated, we cannot draw a conclusion with regard to the effects of untreated S. aureus on the prognosis. In addition, our results suggest that untreated methicillin-resistant CoNS did not worsen morbi-mortality. While the pathogenicity of these microorganisms is well known in several surgical specialities, including orthopaedic, cardio-vascular or neurosurgical cases, the need to treat CoNS in HAI remains unknown. In the current study, 28% of the patients with CoNS samples were not efficiently targeted. These observations illustrate the difficult therapeutic decision for the physician facing CoNS cultures in HAIs. Several hypotheses can explain this observation. The CoNS isolated could be considered as a simple contamination their pathogenicity in this condition might not be important or there could be a synergy between CoNS and other germs that would be suppressed by the treatment of these other germs. Finally, the CoNS could be as pathogenic as S. aureus but not identified due to the insufficient number of patients in this subgroup. This uncertainty leads to suggest targeting these microorganisms in the absence of clinical improvement.

Our study has several limitations. First, it is a retrospective study with inclusions extended over 18 years. However, statistical analysis showed that cases were homogeneously distributed between groups over time, and mortality did not change according to the period of inclusion. Second, many comparisons were performed, which might have increased the probability of type I error. Despite a prolonged duration of inclusion time, we have only collected 29 patients with Sa-HAIs, limiting the value of these observations. Because of the extremely low number of monomicrobial infections, we cannot draw a conclusion about the pathogenicity of staphylococci, especially coagulase-negative staphylococci. It is impossible to differentiate peritoneal colonization from authentic infection. The only clinical surrogates of pathogenic role of the microorganisms cultured from the surgical samples are persistent organ dysfunction and signs of sepsis. This issue leads to cautious consideration of these results and the potential need for antibiotic escalation toward staphylococci for documented therapy in the absence of clinical improvement 21 , 22 . Finally, the pharmacokinetic characteristics of anti-infective agents is another issue to consider. Even if glycopeptide plasma concentrations were monitored on a routine basis, the peritoneal concentrations are impossible to assess, requiring the careful drawing of conclusions regarding the need to target staphylococci.

In summary, the characteristics of HAIs with positive staphylococci peritoneal cultures differ minimally from those without staphylococci, particularly in terms of clinical presentation. Trends toward decreased morbidity criteria were observed in patients with positive staphylococci peritoneal cultures. However, it is not possible, based on such limited signals, to draw conclusions about the absence of the pathogenicity of staphylococci in HAIs or about the lack of need of antibiotic therapy against these microorganisms. In the absence of clinical improvement after adequate source control, antibiotic toward staphylococci should probably be considered if not included in the empirical therapy. Vancomycin has been largely prescribed for enterococci, while at the same time, targeting the peritoneal staphylococci without knowing if they were real pathogens. Evaluating the efficacy of glycopeptides and new anti-Gram-positive molecules, such as linezolid and daptomycin, in the treatment of intra-abdominal infections could be of interest, with the possible restriction of their use against staphylococci.


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