Antibiotic Susceptibility Patterns of Biofilm-Producing Nosocomial Non-Coagulase Staphylococcus Isolates from Clinical Sources in Iraq

Vibrio cholerae, as a natural inhabitant of the marine environment is among the world-leading causes of diarrheal diseases. The present study aimed to investigate the genetic relatedness of Iran 2012–2016 V. cholerae outbreaks with 7th pandemic cholera and to further characterize the non-ST69/non-ST75 sequence types strains by whole-genome sequencing (WGS).Twenty V. cholerae isolates related to 2012, 2013, 2015 and 2016 cholera outbreaks were studied by two genotyping methods – Pulsed-field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST)–and by antimicrobial susceptibility testing. Seven sequence types (STs) and sixteen pulsotypes were detected. Sequence type 69 was the most abundant ST confirming that most (65%, 13/20) of the studied isolates collected in Iran between 2012 and 2016 belonged to the 7th pandemic clone. All these ST69 isolates (except two) exhibited similar pulsotypes. ST75 was the second most abundant ST. It was identified in 2015 and 2016. ST438, ST178, ST579 and STs of 983 and 984 (as newfound STs) each were only detected in one isolate. All strains collected in 2016 appeared as distinct STs and pulsotypes indicative of probable different originations. All ST69 strains were resistant to nalidixic acid. Moreover, resistance to nalidixic acid, trimethoprim-sulfamethoxazole and tetracycline was only observed in strains of ST69. These properties propose the ST69 as a unique genotype derived from a separate lineage with distinct resistance properties. The circulation of V. cholerae ST69 and its traits in recent years in Iran proposes the 7th pandemic strains as the ongoing causes of cholera outbreaks in this country, although the role of ST75 as the probable upcoming dominant ST should not be ignored.Genomic analysis of non-ST69/non-ST75 strains in this study showed ST579 is the most similar ST type to 7th pandemic sequence types, due to the presence of wild type-El Tor sequences of tcpA and VC-1319, VC-1320, VC-1577, VC-1578 genes (responsible for polymyxin resistance in El Tor biotype), the traits of rstC of RS1 phage in one strain of this ST type and the presence of VPI-1 and VSP-I islands in ST579 and ST178 strains. In silico analysis showed no significant presence of resistance genes/cassettes/plasmids within non-ST69/non-ST75 strains genomes. Overall, these data indicate the higher susceptibility of V. cholerae non-ST69/non-ST75 strains in comparison with more ubiquitous and more circulating ST69 and ST75 strains.In conclusion, the occurrence of small outbreaks and sporadic cholera cases due to V. cholerae ST69 in recent years in Iran shows the 7th pandemic strains as the persistent causes of cholera outbreaks in this country, although the role of ST75 as the second most contributed ST should not be ignored. The occurrence of non-ST69/non-ST75 sequence types with some virulence factors characteristics in border provinces in recent years is noteworthy, and further studies together with surveillance efforts are expected to determine their likely route of transport.

To characterize the resistance patterns of uropathogenic Escherichia coli (UPEC) in a Tertiary Teaching Hospital in Iran, we conducted a descriptive epidemiology study using molecular techniques. The subjects consisted of patients having acute urinary tract infection, who were enrolled in the study from 2014 to 2017. The antimicrobial susceptibility profile of 101 UPEC isolates was determined by Kirby-Bauer disc diffusion method. Extended spectrum β-lactamase (ESBL) was detected by the double-disk synergy test. Biofilm formation was done using microtiter plates. The presence of virulence genes (pai, pap, hly, traT, pai, cnf-1, sfa, and afa) was evaluated by a PCR. Molecular typing of UPEC E. coli isolates was performed with fimH and multilocus sequence typing (MLST). 70.3% of isolates were multidrug-resistant. 37.6% of isolates were Extended spectrum β-lactamases (ESBLs) producer. Strong biofilm formation was seen in 27.7%. Forty-seven different fimH allelic variants were identified. Among identified fimH allelic variants, the most common types were f1 (18.8%) and f14 (18.8%). ST131 (54.5%) was the most prevalent clonal group significantly correlated with the pai gene. Seven sequence types (STs) were detected only once (ST405, ST410, ST450, ST636, ST648, ST1193, and ST6451). Clonal groups showed no significant differences in terms of antibiotic resistance patterns. There was no significant difference between virulence genes and antibiotic resistance patterns in the studied clonal groups. To our knowledge, the present study is the first study in Iran that investigated the genotypic diversity of UPEC isolates by MLST and fimH typing methods. The two methods might serve as a useful molecular test for surveillance and epidemiological studies of isolates.

With a high capacity to acquire antimicrobial resistance, Staphylococcus aureus is an important pathogen causing severe infections in animals and humans. A total of 50 Staphylococcus aureus isolates from retail ground beef, chicken meat, and fish were characterized by antimicrobial resistance profiling, staphylococcal protein A gene (spa) typing, and multilocus sequence typing (MLST). The broth microdilution test results showed that all isolates were resistant to penicillin and sulphamethoxazole but had varying resistance rates to tetracycline (24%), erythromycin (4%), gentamicin (2%), ciprofloxacin (2%), trimethoprim (2%), and chloramphenicol (0%). The blaZ and sulI genes were detected in 100% of the isolates followed by grlA (94%), norA (92%), tetK (80%), chlA (60%), tetM (26%), aacA-aphD (2%), ermA (2%), fexA (0%), and dfrA (0%). Moreover, 26% of the isolates were multidrug-resistant, with five or more resistance genes. The spa typing analysis revealed 22 spa types, with t091 (16%), t1677 (8%), and t14538 (8%) being the most common, and one new spa type, t19851, was uncovered. MLST identified seven sequence types (STs), with ST7 (40%), ST15 (20%), and ST199 (13%) being the most common, and two STs (ST7435 and ST7436) were newly identified. In this study, S. aureus isolated from raw meat showed multidrug resistance and different clones associated with human infections. As a result, foods of animal origin may act as potential vehicles for transmission of multidrug-resistant S. aureus isolates, and the dissemination of potentially pathogenic clonal types, posing a health risk to humans.

Multilocus sequence typing (MLST) was performed for vancomycin-resistant Enterococcus faecium (VREF) from diverse geographical areas in Korea to obtain insights into the genetic relationships with other molecular profiles. To understand the diversity of lineages, vancomycin-susceptible E. faecium (VSEF) were included. A total of 60 E. faecium isolates were analysed by MLST and esp profile. Molecular typing of Tn1546 of 30 VREF strains was evaluated by overlapping PCR of Tn1546 and DNA sequencing. Seven sequence types (ST) were found among 30 VSEF isolates, and four STs were found among 30 VREF isolates. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Of the 60 E. faecium isolates, 35 isolates were positive for the esp gene. On molecular typing of Tn1546, all VREF isolates were divided into four main types. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type represented different STs. An association between Tn1546 typing and MLST was not found. These results suggest that the horizontal spread of Tn1546 between strains plays a major role in the dissemination of vancomycin resistance in Korea.

In this study, 644 raw milk samples were collected from various milk sources of Khartoum state during May 2003 till April 2004. Using the API kits, both coagulase positive and negative Staphylococci (CPS and CNS) were identified. Most of the CPS isolates were Staphylococcus aureus, while the majority of CNS wereStaphylococcus epidermidis. 23.8% of the samples were found to be positive to both CPS and CNS while 33.7% of the samples were negative to both CPS and CNS. Forty one percent of CPS isolates was found to be S. aureus and 39% of CNS were S. epidermidis. The coagulase positive Staphylococci (CPS) were determined in 45% of the tested samples while coagulase negative Staphylococci(CNS) were found in 44.7% of them, of which 16.5% exceeded the recommended bacterial safety limits for CPS. The highest of the samples that exceeded this limit was found in vendor milk samples (19.4%) while the market milk was 18.4%. Key words: Staphylococci, bovine, raw, milk, Sudan.

Stenotrophomonas maltophilia is an opportunistic nosocomial pathogen and is being isolated worldwide with increasing frequency. S. maltophilia isolates are often highly resistant to most of the currently used antimicrobial agents, including carbapenems, aminoglycosides, and fluoroquinolones. A variety of antimicrobial resistance mechanisms, including efflux pumps and integrons, have been reported in S. maltophilia. In Korea, the isolation rate of S. maltophilia is increasing, but few studies have reported the resistance mechanisms of this pathogen. Cho et al. [1] reported the first study on efflux pumps in clinical isolates of S. maltophilia in Korea. Efflux pumps have been shown to play a role in multidrug resistance in Pseudomonas aeruginosa, Burkholderia cepacia, and S. maltophilia. The findings of the study by Cho et al. have indicated that overexpression of SmeABC efflux pumps is important for the fluoroquinolone (ciprofloxacin and levofloxacin) resistance of S. maltophilia. The conclusions of this study are interesting from an epidemiological perspective. Multilocus sequence typing (MLST) has recently been used to type many pathogenic bacteria and fungi. MLST has many advantages over other molecular typing methods; it is easy to perform and produces unambiguous results, and the sequence data can be shared and compared among different hospitals [2]. However, to date, MLST has seldom been applied for typing clinical S. maltophilia isolates. The report by Kaiser et al. [3] described the development of an MLST scheme for S. maltophilia. They applied MLST to S. maltophilia based on partial sequences (444 to 558 bp) of 7 housekeeping genes. The online database (http://pubmlst.org/smaltophilia/) includes 40 to 55 alleles for each of the 7 target gene sequences and 56 multilocus allelic profiles or sequence types (ST), which is considerably lower than the numbers deposited in databases for other bacteria. Cho et al. [1] used MLST to study the molecular epidemiology of S. maltophilia at a university hospital in Korea. The 33 clinical isolates recovered over a 1-yr period exhibited 26 different STs. Of these, 28 isolates exhibited new STs (23 distinct STs), whereas only 5 isolates exhibited previously described STs (3 STs). Their study proved that the MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and that it is appropriate for studying population structures. The genetic diversity of the S. maltophilia isolates in the study by Cho et al. indicates that most of these resistant bacteria may be of a polyclonal nature. However, the authors have emphasized on the clonal nature of the emergence of antimicrobial-resistant S. maltophilia, as shown in a phylogenetic analysis of MLST types. Three or 4 isolates showing a high level of resistance to ciprofloxacin and overexpression of the SmeABC efflux system were observed in groups A and A' of the MLST cluster, respectively. Additionally, the group B isolates (4 isolates) were highly resistant to ceftazidime and cefepime. This finding suggests that MLST data can provide evidence of the emergence and spread of antimicrobial-resistant clones of S. maltophilia. Molecular epidemiological studies provide important tools to prevent the further spread of antimicrobial-resistant nosocomial pathogens. MLST can be used as an alternative to pulsed-field gel electrophoresis (PFGE) in clinical microbiology. Many studies have suggested that these techniques have similar powers of discrimination for several pathogens, but this discriminatory power differs among species. MLST generally provides information on the evolutionary history, population structure, and longterm global epidemiology of pathogens, whereas PFGE is better for investigation of outbreaks and potential transmission events over a short period of time [2]. Therefore, after further validation and comparison with PFGE by using a larger collection of isolates, MLST may play a role in clinical microbiology by facilitating the investigation of molecular epidemiology of S. maltophilia strains. The present MLST analysis provides valuable data regarding the current epidemic status of antimicrobial-resistant S. maltophilia isolates and their antimicrobial efflux mechanisms under hospital settings. The increase in the prevalence of nosocomial S. maltophilia infections is principally because of the changing characteristics of hospitalized patients and use of antibiotics. In this regard, the work of Cho et al. is important because it highlights the fact that MLST is a useful method to elucidate the trends of resistance in S. maltophilia isolates associated with nosocomial infections. Moreover, this technique will facilitate identification of the clonality and evolutionary relatedness of antimicrobial-resistant S. maltophilia isolates.

The increasing prevalence of Staphylococcus haemolyticus infections in community and hospital settings presents a significant health challenge due to growing antibiotic resistance and biofilm formation. This study aims to:(1) perform a molecular analysis of prevalent native strains in Anbar, Iraq, (2) differentiate between various pathogenic strains using multilocus sequence typing (MLST) to enhance epidemiological and surveillance efforts. The objective is to trace the origins of these strains and distinguish between invasive and indigenous strains. While S. haemolyticus is generally part of the normal human microbiota, it can lead to serious infections in individuals with prior injuries or surgical procedures. It is particularly skilled at developing antibiotic resistance, making it a leading cause of hospital-acquired infections, largely through the staphylococcal cassette chromosome mec (SCCmec). Methicillin-resistant S. haemolyticus (MRSH) has developed resistance to oxacillin/cefoxitin through SCCmec acquisition, and hospital-associated MRSH strains are increasingly resistant to multiple antibiotics. The preparation of blood agar medium followed the manufacturer's guidelines. After autoclaving at 121ºC for 15 minutes, the medium was cooled to 50ºC. The mixture was then thoroughly mixed and poured into sterile Petri dishes. This medium is used for isolating and cultivating bacteria, as well as for detecting hemolytic activity and identifying the type of hemolysis. Genomic extraction and molecular screening of multidrug- resistant (MDR) isolates were performed, followed by MLST analysis. Data were processed using the University of Nebraska Medical Center's pubMLST website. To explore the genetic relationships among S. haemolyticus strains, their genomic DNA was analyzed using MLST typing based on the protocol from the MLST Institute database. All S. haemolyticus isolates in the study underwent MLST gene screening through PCR to verify the presence of housekeeping genes (arc, SH1200, hemH, leuB, SH1341, cfxE, and ribose ABC). PCR electrophoresis results demonstrated successful amplification of all target genes, confirming their appropriateness for MLST analysis. Three isolates were recognized as novel global strains, designated ST153, ST154, and ST155. In addition, five other strains were previously registered as ST3, ST9, ST29, ST123, and ST124. The findings diverge from the established global understanding of type distribution in Asia. To combat the spread of highly resistant strains, it is crucial to monitor virulence factors and antibiotic resistance closely.

Pathogen typing in the genomics era: MLST and the future of molecular epidemiology

Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins

Vibrio cholerae O1 strains taken from the repository of Yunnan province, southwest China, were abundant and special. We selected 70 typical toxigenic V. cholerae (69 O1 and one O139 serogroup strains) isolated from Yunnan province, performed the pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and MLST of virulence gene (V-MLST) methods, and evaluated the resolution abilities for typing methods. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. Seventy V. cholerae obtained 50 PFGE patterns, with a high resolution. The strains could be divided into three groups with predominance of strains isolated during 1980s, 1990s, and 2000s, respectively, showing a good consistency with the epidemiological investigation. We also evaluated two MLST method for V. cholerae, one was used seven housekeeping genes (adk, gyrB, metE, pntA, mdh, purM, and pyrC), and all the isolates belonged to ST69; another was used nine housekeeping genes (cat, chi, dnaE, gyrB, lap, pgm, recA, rstA, and gmd). A total of seven sequence types (STs) were found by using this method for all the strains; among them, rstA gene had five alleles, recA and gmd have two alleles, and others had only one allele. The virulence gene sequence typing method (ctxAB, tcpA, and toxR) showed that 70 strains were divided into nine STs; among them, tcpA gene had six alleles, toxR had five alleles, while ctxAB was identical for all the strains. The latter two sequences based typing methods also had consistency with epidemiology of the strains. PFGE had a higher resolution ability compared with the sequence based typing method, and MLST used seven housekeeping genes showed the lower resolution power than nine housekeeping genes and virulence genes methods. These two sequence typing methods could distinguish some epidemiological special strains in local area.

Temporal changes in the genotypes of methicillin-resistant Staphylococcus aureus strains isolated from a tertiary Malaysian hospital based on MLST, spa, and mec-associated dru typing

Cryptococcus species is an opportunistic yeast pathogen and classified into different molecular types according to typing techniques including multilocus sequence typing (MLST). The study aimed to investigate the genotypes of environmental Cryptococcus isolates using MLST and the relationship between the in vitro antifungal susceptibility and sequence types of isolates. Genotyping Cryptococcus isolates was performed by the MLST method at seven nuclear loci. Antifungal susceptibility was determined by using CLSI broth micro-dilution method for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine, and luliconazole. Seven sequence types (ST) were detected using MLST analysis, with the most frequent (50%) ST77, followed by ST4 (16.7%) among 30 C. neoformans isolates. All antifungals demonstrated excellent activity against isolates, except for itraconazole and amphotericin B that were non-wild type against 53.3% and 10% of isolates, respectively. Although seven sequence types belonging to C. neoformans isolates were detected, ST77 was the main sequence type in Ahvaz. Also, non-wild type isolates were only found against itraconazole and amphotericin B.

Staphylococci involve infections in association with a number of bacterial virulence factors. Extracellular enzymes play an important role in staphylococcal pathogenesis. In addition, biofilm is known to be associated with their virulence. In this study, 149 staphylococcal isolates from acne lesions were investigated for their virulence factors including lipase, protease, and biofilm formation. Coagulase-negative staphylococci were demonstrated to present lipase and protease activities more often than coagulase-positive staphylococci. A microtiter plate method (quantitative method) and a Congo red agar method (qualitative method) were comparatively employed to assess biofilm formation. In addition, biofilm forming ability was commonly detected in a coagulase-negative group (97.7%, microtiter plate method and 84.7%, Congo red agar method) more frequently than in coagulase-positive organisms (68.8%, microtiter plate method and 62.5%, Congo red agar method). This study clearly confirms an important role for biofilm in coagulasenegative staphylococci which is of serious concern as a considerable infectious agent in patients with acnes and implanted medical devices. The Congo red agar method proved to be an easy method to quickly detect biofilm producers. Sensitivity of the Congo red agar method was 85.54% and 68.18% and accuracy was 84.7% and 62.5% in coagulase-negative and coagulase-positive staphylococci, respectively, while specificity was 50% in both groups. The results clearly demonstrated that a higher percentage of coagulasenegative staphylococci isolated from acne lesions exhibited lipase and protease activities, as well as biofilm formation, than coagulase-positive staphylococci.

Investigations of the population genetics of Bartonella henselae have demonstrated a high level of diversity among strains, and the delineation of isolates into one of two subtypes, type I (Houston) and type II (Marseille), represented by specific 16S ribosomal DNA (rDNA) sequences, has long been considered the most significant genotypic division within the species. This belief is challenged by recent work suggesting a role for horizontal gene exchange in generating intraspecies diversity. We attempted to resolve this issue and extend exploration of the population structure of B. henselae by using multilocus sequence typing (MLST) to examine the distribution of polymorphisms within nine different genes in a sample of 37 human and feline isolates. MLST distinguished seven sequence types (STs) that resolved into three distinct lineages, suggesting a clonal population structure for the species, and support for these divisions was obtained by macrorestriction analysis using pulsed-field gel electrophoresis. The distribution of STs among isolates recovered from human infections was not random, and such isolates were significantly more often associated with one particular ST, lending further support to the suggestion that specific genotypes contribute disproportionately to the disease burden in humans. All but one isolate lay on lineages that bore the representative strain of either the Houston or Marseille subtype. However, the distribution of the two 16S rDNA alleles among the isolates was not entirely congruent with their lineage allocations, indicating that this is not a sensitive marker of the clonal divisions within the species. The inheritances of several of the genes studied could not be reconciled with one another, providing further evidence of horizontal gene transfer among B. henselae strains and suggesting that recombination has a role in shaping the genetic character of bartonellae.

Campylobacteriosis is the most frequent zoonosis in developed countries and various domestic animals can function as reservoir for the main pathogens Campylobacter jejuni and Campylobacter coli. In the present study we compared population structures of 730 C. jejuni and C. coli from human cases, 610 chicken, 159 dog, 360 pig and 23 cattle isolates collected between 2001 and 2012 in Switzerland. All isolates had been typed with multi locus sequence typing (MLST) and flaB-typing and their genotypic resistance to quinolones was determined. We used complementary approaches by testing for differences between isolates from different hosts with the proportion similarity as well as the fixation index and by attributing the source of the human isolates with Bayesian assignment using the software STRUCTURE. Analyses were done with MLST and flaB data in parallel and both typing methods were tested for associations of genotypes with quinolone resistance. Results obtained with MLST and flaB data corresponded remarkably well, both indicating chickens as the main source for human infection for both Campylobacter species. Based on MLST, 70.9% of the human cases were attributed to chickens, 19.3% to cattle, 8.6% to dogs and 1.2% to pigs. Furthermore we found a host independent association between sequence type (ST) and quinolone resistance. The most notable were ST-45, all isolates of which were susceptible, while for ST-464 all were resistant.