Spread of an OmpK36-modified ST15 Klebsiella pneumoniae variant during an outbreak involving multiple carbapenem-resistant Enterobacteriaceae species and clones

We aim to characterise multiple ertapenem-resistant (ERT-R, n?=?15) Enterobacteriaceae isolates identified as presumptive carbapenemase producers in a Portuguese hospital in a short period of time (March–July 2010). Antibiotic susceptibility patterns, β-lactamases, genetic relatedness [pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST)], plasmid content and major enterobacterial porins were investigated. Ertapenem resistance was associated with deficiencies in major porins and, in some cases, extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase production among outbreak and non-outbreak clones. Most isolates (n?=?8) corresponded to two ERT-R Klebsiella pneumoniae ST15 PFGE-types: (i) a sporadic variant (Kp-A-ERT, n?=?1) presenting a premature stop codon in ompK36 and (ii) an epidemic variant (Kp-B-ERT, n?=?7) exhibiting a new OmpK36 porin variant, which differed additionally in plasmid and antibiotic susceptibility profiles. ST14 (n?=?1) and ST45 (n?=?1) K. pneumoniae, ST131 (n?=?1) and ST354 (n?=?1) Escherichia coli, Enterobacter asburiae (n?=?1), Enterobacter cloacae (n?=?1) and Enterobacter aerogenes (n?=?1) ERT-R clones were also sporadically detected. Porin changes in these isolates included non-sense mutations [ompK35, ompK36, ompF; minimum inhibitory concentration (MIC)?=?4–32?mg/l], IS-mediated porin disruptions (ompK36, ompC; MIC?=?12–>32?mg/l) or alterations in the L3 loop (ompK36; MIC?=?4–16?mg/l). We describe, for the first time in Portugal, the simultaneous emergence of multiple ERT-R Enterobacteriaceae species and clones in a short period of time. Moreover, our results support that a CTX-M-15-producing ST15 K. pneumoniae with an OmpK36-modified porin might successfully spread in the nosocomial setting.     

Molecular Characterisation for Clonality and Transmission Dynamics of an Outbreak of Klebsiella pneumoniae amongst Neonates in a Tertiary Care Centre in South India

Purpose: Sepsis is a significant cause of morbidity and mortality amongst neonates. Klebsiella pneumoniae is a common cause of nosocomial outbreaks causing bacteraemia and having potential of acquiring plasmids enhancing antimicrobial resistance. In the present study, we investigate K. pneumoniae outbreak causing bacteraemia amongst neonates over a span of 2 months. Isolates were characterised for antimicrobial resistance, virulence, molecular typing for clonality and plasmid typing for transmission dynamics, and patient outcome was investigated. Methods: Thirteen isolates of K. pneumoniae were obtained during October–November 2016. Antimicrobial susceptibility testing was performed, and multiplex polymerase chain reaction (PCR) for β-lactamases and PCR for ompK35 and ompK36 were performed. To study hypervirulence, string test and PCR for rmpA and rmpA2 were performed. Multilocus sequence typing and Inc plasmid typing were carried out to study transmission dynamics. Results: Amongst 13 isolates, all isolates harboured bla_SHV and bla_TEM; 12 isolates carried bla_CTX-M-1. ompK35 was present in all, but ompK36 was absent in 12 isolates. Ten isolates belonged to ST48, 6 amongst which contained IncFII (K) plasmid. One isolate each belonged to ST29, ST111 and ST2647 (novel clone). None of the isolates was hypervirulent. Conclusion: Extended-spectrum β-lactamase K. pneumoniae is commonly seen in Indian hospitals and main mechanisms being production of SHV, TEM and CTX-M enzymes as seen in the present study. Outer membrane porins contribute significantly to antimicrobial resistance. Emergence of new clones such as ST2647 implies continuous evolution of the organism and also potential for rapid genetic recombination leading to multidrug resistance. Outbreaks amongst neonates lead to fatal outcome, and stringent hospital infection control is necessary.     

Capsular serotypes and multilocus sequence types of bacteremic Klebsiella pneumoniae isolates associated with different types of infections

We investigated the epidemiology of different serotypes of Klebsiella pneumoniae isolates causing bacteremic liver abscess (LA) using multilocus sequence typing (MLST). MLST and molecular typing were performed for 41 K1 (19 LA), 37 K2 (5 LA), and 33 non-K1/K2 (6 LA) isolates that were derived from a previous one-year K. pneumoniae bacteremia cohort. Capsular serotypes and rmpA of these isolates were determined by polymerase chain reaction (PCR) methods. Among the 41 K1 isolates, 39 were ST23 and the remaining two isolates were ST23 single-locus variant. There were 11 STs among K2 isolates. ST65 was the most common (n?=?10), followed by ST86, ST373, and ST375. Only ST65 (n?=?3), ST373 (n?=?1), and ST375 (n?=?1) caused LA, and ST65 was a three-locus variant of ST23. For non-K1/K2 isolates, the ST types varied widely. ST218 (K57) was the most common type (n?=?6, 18 %), and it was a single-locus variant of ST23 and caused two cases of LA. The existences of rmpA among serotypes varied (100 % for K1, 89 % for K2, and 55 % for non-K1/K2). For isolates causing LA, all of them were positive for rmpA. For non-K1/K2 isolates causing infections other than LA, the positivity of rmpA ranged from 0 % (biliary tree infection) to 67 % (pneumonia). In this one-year cohort, all K1 isolates were ST23 or its single-locus variants, but the composition of ST types among K2 isolates was quite variable. ST23 and its one- (ST1005 and ST218) and three-locus (ST65) variants comprised 80 % of isolates causing LA.     

Evaluation of ompK36 allele groups on clinical characteristics and virulence features of Klebsiella pneumoniae from bacteremia

Background/purposeThis study investigated the implications of ompK36 allele groups on clinical and microbiological features of patients with Klebsiella pneumoniae bacteremia.MethodsA total of 80 K. pneumoniae bloodstream isolates were collected and then divided into four ompK36 allele groups. Clinical characteristics, bacterial antibiotic resistance and virulence determinants were analyzed, including resistance and virulence genes, hypermucoviscosity phenotype, K capsule serotypes, biofilm formation, serum killing, neutrophil phagocytosis, and mouse lethality studies.Results78 isolates were classified into four ompK36 variants, designated groups A (34), B (6), C (26), and D (12), respectively; 2 isolate was untypeable. OmpK36 group C isolates carried higher frequencies of K1/K2 capsule serotypes, hypermucoviscosity phenotype, rmpA gene, allS gene, iroB gene, aerobactin gene, or rmpA2 gene than non-C group isolates. OmpK36 group C isolates were significantly more virulent, as higher serum resistance, higher anti-phagocytosis and higher mouse lethality, than OmpK36 non-C group isolates, except for similar biofilm formation capability. The K20 isolates probably has low expression rates of rmpA and rmpA2 for hypermucoviscosity phenotype. The biofilm formation was significantly associated with ESBL production. OmpK36 group C isolates were more frequently detected in patients with community-acquired bloodstream infection. However, significant underlying diseases and prior use of carbapenem were highly prevalent in patients with OmpK36 non-C group isolates infection. ESBL production was apparently higher in non-C group but did not reach statistical significance.ConclusionOur results suggest that the OmpK36 group C K.pneumoniae is more associated with community-acquired infection with a lower frequency of underlying illness, but with significantly more virulence in bloodstream infection. This would give a remind that clinicians should be aware of such clinical impacts of the ompK36 allele group.     

Predictability of Phenotype in Relation to Common β-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae

The minimal concentration of antibiotic required to inhibit the growth of different isolates of a given species with no acquired resistance mechanisms has a normal distribution. We have previously shown that the presence or absence of transmissible antibiotic resistance genes has excellent predictive power for phenotype. In this study, we analyzed the distribution of six β-lactam antibiotic susceptibility phenotypes associated with commonly acquired resistance genes in Enterobacteriaceae in Sydney, Australia. Escherichia coli (n = 200) and Klebsiella pneumoniae (n = 178) clinical isolates, with relevant transmissible resistance genes (bla_TEM, n = 33; plasmid AmpC, n = 69; extended-spectrum β-lactamase [ESBL], n = 116; and carbapenemase, n = 100), were characterized. A group of 60 isolates with no phenotypic resistance to any antibiotics tested and carrying none of the important β-lactamase genes served as comparators. The MICs for all drug-bacterium combinations had a normal distribution, varying only in the presence of additional genes relevant to the phenotype or, for ertapenem resistance in K. pneumoniae, with a loss or change in the outer membrane porin protein OmpK36. We demonstrated mutations in ompK36 or absence of OmpK36 in all isolates in which reduced susceptibility to ertapenem (MIC, >1 mg/liter) was evident. Ertapenem nonsusceptibility in K. pneumoniae was most common in the context of an OmpK36 variant with an ESBL or AmpC gene. Surveillance strategies to define appropriate antimicrobial therapies should include genotype-phenotype relationships for all major transmissible resistance genes and the characterization of mutations in relevant porins in organisms, like K. pneumoniae.     

Antimicrobial resistance comparison of Klebsiella pneumoniae pathogens isolated from intra-abdominal and urinary tract infections in different organs,hospital departments and regions of China between 2014 and 2017

BackgroundWe describe antibiotic resistance trends of Klebsiella pneumoniae pathogens, responsible for urinary tract infections (UTIs) and intra-abdominal infections (IAIs), isolated from different organs and tissues, hospital departments and Chinese regions between 2014 and 2017.MethodsResistances of UTIs and IAIs derived K. pneumoniae isolates from 17 hospitals in 7 Chinese regions to amikacin, imipenem, piperacillin-tazobactam, ertapenem, and cefepime were unequivocally established.ResultsOverall resistance rates of K. pneumoniae IAI isolates obtained from gallbladder and abscesses increased to amikacin (14.29–30.95%) and for liver, gallbladder, and abscesses to imipenem (14.29–38.10%), piperacillin-tazobactam (23.81–38.10%), and ertapenem (23.81–38.10%) in 2017, but were constant (20–30%) for K. pneumoniae isolates from UTIs from 2014 to 2017. In medical and surgical ICUs, resistance rates to all tested antibiotics rose to ∼60% for IAIs, which was also reflected in higher resistance rates of hospital acquired (HA) compared to community acquired (CA) infections. In medical ICUs resistance rates increased to 50–60% for amikacin, imipenem, and ertapenem for UTI-derived K. pneumoniae isolates in 2017. Resistance rates to all tested antibiotics were highest in the east Jiangzhe region of China, being ∼60% for K. pneumoniae isolates from IAIs and 40% for K. pneumoniae isolates from UTIs to ertapenem and imipenem, as well as > 40% for piperacillin-tazobactam in 2017.ConclusionIn China, ICUs resistance rates to K. pneumoniae IAIs and UTIs isolates was increased in 2017 for all tested antimicrobials including carbapenems, which makes them no longer suitable for empiric treatment. In the east Jiangzhe region this was a general trend that was independent of the type of hospital department.     

Outbreak Caused by an Ertapenem-Resistant,CTX-M-15-Producing Klebsiella pneumoniae Sequence Type 101 Clone Carrying an OmpK36 Porin Variant

Although numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae (CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum β-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining β-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the bla_CTX-M-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniae isolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak.     

Prevalence and Characteristics of the Epidemic Multiresistant Escherichia coli ST131 Clonal Group among Extended-Spectrum Beta-Lactamase-Producing E. coli Isolates in Copenhagen,Denmark

We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates (n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH, fyuA, malX, and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously.     

The Resistance Mechanism and Clonal Distribution of Tigecycline-Nonsusceptible Klebsiella pneumoniae Isolates in Korea

PurposeTigecycline is one of the drugs used to treat multi-drug resistant Klebsiella pneumoniae (K. pneumoniae) infections, including complicated skin and soft tissue infections, complicated intra-abdominal infection, and community-acquired pneumonia in the Republic of Korea. However, since its commercial release, K. pneumoniae resistance against tigecycline has been reported, and there is a serious concern about the spread of tigecycline resistant bacteria.ResultsFifty-six K. pneumoniae isolates were found to be nonsusceptible, 16% of the 342 collected. Twenty-seven and nine isolates of the tigecycline nonsusceptible isolates had mutations in the ramR and rpsJ genes, respectively; while 18 nonsusceptible isolates harbored the tetA gene. Comparison of isolates with and without ramR mutation showed a significant statistical difference (p<0.05) for expression of AcrAB. Moreover, the most common clonal types, as observed in our study, appear to be ST11 and ST789.ConclusionSeveral dominate clonal types infer tigecycline resistance to K. pneumoniae, including ST11, ST768, ST15, ST23, ST48, and ST307. There does not seem to be a transferrable medium, such as plasmid, for the resistance yet, although mutation of the ramR gene may be a common event, accounting for 48% of the nonsusceptibility in this study.     

Molecular epidemiology and resistance patterns of blaOXA-48 Klebsiella pneumoniae and Escherichia coli: A nationwide multicenter study in Taiwan

BackgroundWe describe the molecular epidemiology and resistance patterns of bla_OXA-48 Klebsiella pneumoniae and Escherichia coli in Taiwan.MethodsIn this multicenter surveillance study from January 2012 to August 2015, the identified bla_OXA-48 Enterobacteriaceae isolates were subjected to antibiotics susceptibility testing. PCR method was used for detecting concomitant other beta-lactamases. Outer membrane porins were analyzed. Genetic relatedness and molecular epidemiology of the isolates were determined through pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid incompatibility was determined using PCR-based replicon typing.ResultsForty-three bla_OXA-48 K. pneumoniae and two E. coli isolates were analyzed. The annual incidence of bla_OXA-48 K. pneumoniae isolates from 2012 to 2015 were 0%, 1.1%, 2.4%, and 7.6%, respectively. Forty-three (95.5%) of 45 isolates were non-susceptible to broad-spectrum beta-lactams (ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam), Forty-two (93.3%) of the 45 isolates showed resistance against all tested carbapenems (imipenem, meropenem, doripenem, and ertapenem). Molecular characterization revealed that they co-produced at least one extended-spectrum beta-lactamases or AmpC beta-lactamases, with at least one outer membrane porin loss. Thirty-eight (88.3%) of the 43 K. pneumoniae isolates belonged to ST11. PFGE analysis of 43 K. pneumoniae isolates revealed dissemination of multiple clones. Six of the 12 tested K. pneumoniae representatives of different pulso-types belonged to IncA/C.ConclusionConcomitant loss of porins and production of other beta-lactamases renders the bla_OXA-48-producing isolates in Taiwan a high-level carbapenem resistance and broad resistance against many beta-lactam antibiotics. Following dissemination of multiple clones of bla_OXA-48 K pneumoniae ST 11, a trend of increased bla_OXA-48 prevalence was noted.     

Increasing incidence of carbapenemase-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in Belgian hospitals

Carbapenemase-producing Enterobacteriaceae are increasingly reported worldwide. The aim of the study was to determine the incidence and molecular epidemiology of carbapenemase-producing (CP) Escherichia coli and Klebsiella pneumoniae (CP-E/K) in Belgium. Eleven hospital-based laboratories collected carbapenem non-susceptible (CNS) isolates of E. coli and K. pneumoniae detected in clinical specimens from January 2013 to December 2014. All CNS strains were tested for carbapenemase production and typed by multilocus sequence typing (MLST) for a 6-month period as part of the European Survey on Carbapenemase-Producing Enterobacteriaceae in Europe (EuSCAPE) structured survey. In addition, an equal number of carbapenem-susceptible isolates collected were preserved as a control group for risk factor analysis. The overall incidence rate of CP-E/K isolates in hospitals increased from 0.124 in 2013 to 0.223 per 1000 admissions in 2014. From November 2013 to April 2014, 30 CP K. pneumoniae [OXA-48 (n?=?16), KPC (n?=?13), OXA-427 (n?=?1)] and five CP E. coli [OXA-48 (n?=?3), NDM (n?=?1), OXA-427 (n?=?1)] isolates were detected in ten hospitals. The 16 OXA-48-producing K. pneumoniae strains were distributed into eight sequence types (STs), while the 13 KPC-producing K. pneumoniae clustered into three STs dominated by ST512 (n?=?7) and ST101 (n?=?5). Compared to controls, we observed among CP-E/K carriers significantly higher proportion of males, respiratory origins, previous hospitalization, nosocomial setting, and a significantly lower proportion of bloodstream infections. Our study confirms the rapid spread of CP-E/K in Belgian hospitals and the urgent need for a well-structured and coordinated national surveillance plan in order to limit their dissemination.     

Fitness cost associated with resistance to fluoroquinolones is diverse across clones of Klebsiella pneumoniae and may select for CTX-M-15 type extended-spectrum β-lactamase

Lowered fitness cost associated with resistance to fluoroquinolones was recently demonstrated to influence the clonal dynamics of methicillin-resistant Staphylococcus aureus (MRSA) in the health care setting. We investigated whether or not a similar mechanism impacts Klebsiella pneumoniae. The fitness of K. pneumoniae isolates from major international hospital clones (ST11, ST15, ST147) already showing high-level resistance to fluoroquinolones and of strains from three minor clones (ST25, ST274, ST1028) in which fluoroquinolone resistance was induced in vitro was tested in a propagation assay. Strains from major clones showed significantly less fitness cost than three of four fluoroquinolone-resistant derivatives of minor clone isolates. In addition, plasmids with CTX-M-15 type extended-spectrum β-lactamase (ESBL) genes were all retained in both major and minor clone isolates, irrespective of the strains’ level of fluoroquinolone resistance, while each plasmid harboring SHV-type ESBLs had been lost during the induction of resistance. Major clone K. pneumoniae strains harbored more amino acid substitutions in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes than minor clone isolates. The presence of an active efflux system could be demonstrated in all fluoroquinolone-resistant derivatives of originally SHV-producing minor clone isolates but not in any CTX-M-15-producing strain. Further investigations are needed to expand and confirm our findings on a larger sample. In addition, a long-term observation of our ciprofloxacin-resistant minor clone isolates is required in order to elucidate whether or not they are capable of restoring their fitness while concomitantly retaining high minimum inhibitory concentration (MIC) values.     

A nosocomial outbreak of KPC-2-producing Klebsiella pneumoniae in a Chinese hospital: dissemination of ST11 and emergence of ST37, ST392 and ST395

In China, Klebsiella pneumoniae carbapenemase (KPC) -producing K. pneumoniae isolates have been identified. However, little is known about the spread and outbreak of KPC-producing enterobacterial pathogens. In this study, 48 non-duplicated KPC-producing isolates were analysed for genetic relatedness by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility by E-test, and sequence type (ST) by multilocus sequence typing. S1-PFGE and Southern blot were used for plasmid profiling, and PCR and subsequent sequencing were performed to determine the effects of genetic background on the blaKPC gene. From December 2011 to June 2012, an outbreak of the KPC-2-producing K. pneumoniae was observed. The 48 isolates of K. pneumoniae are categorized into eight PFGE types (A1, A2, A3, A4, B, C, D and E). The predominant pathogens of the outbreak were strains with PFGE types A1, A2 and A3, which all belong to ST11. Furthermore, ST37, ST392 and ST395 KPC-2-producing K. pneumoniae isolates have also been sporadically identified. The blaKPC-2-carrying plasmids vary in size from 30 to 220 kb. The genetic environments of the blaKPC-2 gene for most strains were consistent with the genetic structure of blaKPC-2 on the plasmid pKP048. In conclusion, the dissemination and outbreak of KPC-2-producing K. pneumoniae isolates in this study appeared to be clonal, and ST11 K. pneumoniae was the predominant clone attributed to the outbreak. This is the first study to report the emergence and spread of KPC-producing K. pneumoniae ST392 and ST395 worldwide. Our findings suggest that horizontal transfer of Tn3-based transposons might mediate the spread of blaKPC-2 gene between different K. pneumoniae clones in China.     

Outbreak of KPC-3-producing,and colistin-resistant,Klebsiella pneumoniae infections in two Sicilian hospitals

We report the first outbreak caused by colistin-resistant Klebsiella pneumoniae producing KPC-3 carbapenamase in two Italian hospitals. This spread occurred in 1 month, and was caused by eight colistin-resistant and carbapenem-resistant Klebsiella pneumoniae isolates from eight patients. A further three isolates were obtained from the intestinal tract and pharyngeal colonization. All isolates were multidrug-resistant (MDR), including being resistant to colistin, but they were susceptible to gentamicin and tigecycline. PCR detection showed that all isolates harboured the bla_KPC-3 gene associated with bla_SHV-11, bla_TEM-1 and bla_OXA-9. All K. pneumoniae isolates, genotyped by pulsed-field gel electrophoresis and multilocus sequence typing, belonged to the same sequence type (ST)258 clone. From our data and a review of the international literature, K. pneumoniae ST258 seems to be the most widespread genetic background for KPC dissemination in Europe.     

Clonal dissemination of multilocus sequence type ST15 KPC‐2‐producing Klebsiella pneumoniae in Bulgaria

A total of 36 consecutive clinical and two fecal‐screening carbapenem‐resistant Klebsiella pneumoniae isolates from two Bulgarian university hospitals (Varna and Pleven) were investigated. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta‐lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and multilocus sequence typing (MLST). Most of the isolates demonstrated multidrug resistance profile. Amikacin and tigecycline retained good activity with susceptibility rates of 95 and 87%, respectively. The resistance rate to colistin was 63%. Six RAPD‐ and MLST‐types were identified: the dominating MLST‐type was ST15 (27 isolates), followed by ST76 (six isolates), and ST1350 (two isolates). ST101, ST258, and ST151 were detected once. All except one of the K. pneumoniae produced KPC‐2, mostly in combination with CTX‐M‐15, while for one isolate (ST101) the enzymes OXA‐48 and CTX‐M‐14 were found. All KPC‐2‐producing transconjugants revealed the presence of IncFII plasmid. The OXA‐48‐ and CTX‐M‐14‐producing isolate showed the presence of L/M replicon type. The dissemination of KPC‐2‐producing K.pneumoniae in Bulgaria is mainly due to the sustained spread of successful ST15 clone and to a lesser extent of ST76 clone. This is the first report of OXA‐48 producing ST101 K. pneumoniae in Bulgaria.     

Potential virulence of Klebsiella sp. isolates from enteral diets

We aimed to evaluate the potential virulence of Klebsiella isolates from enteral diets in hospitals, to support nosocomial infection control measures, especially among critical-care patients. Phenotypic determination of virulence factors, such as capsular expression on the external membrane, production of aerobactin siderophore, synthesis of capsular polysaccharide, hemolytic and phospholipase activity, and resistance to antibiotics, which are used therapeutically, were investigated in strains of Klebsiella pneumoniae and K. oxytoca. Modular industrialized enteral diets (30 samples) as used in two public hospitals were analyzed, and Klebsiella isolates were obtained from six (20%) of them. The hypermucoviscous phenotype was observed in one of the K. pneumoniae isolates (6.7%). Capsular serotypes K1 to K6 were present, namely K5 and K4. Under the conditions of this study, no aerobactin production, hemolytic activity or lecithinase activity was observed in the isolates. All isolates were resistant to amoxicillin and ampicillin and sensitive to cefetamet, imipenem, chloramphenicol, gentamicin and sulfamethoxazole-trimethoprim. Most K. pneumoniae isolates (6/7, 85.7%) from hospital B presented with a higher frequency of resistance to the antibiotics tested in this study, and multiple resistance to at least four antibiotics (3/8; 37.5%) compared with isolates from Hospital A. The variations observed in the antibiotic resistance profiles allowed us to classify the Klebsiella isolates as eight antibiotypes. No production of broad-spectrum β-lactamases was observed among the isolates. Our data favor the hypothesis that Klebsiella isolates from enteral diets are potential pathogens for nosocomial infections.     

Escherichia coli Sequence Type 131 as a Prominent Cause of Antibiotic Resistance among Urinary Escherichia coli Isolates from Reproductive-Age Women

The recent emergence of multidrug-resistant Escherichia coli sequence type 131 (ST131) has coincided with an increase in general antibiotic resistance of E. coli, suggesting that ST131 has a contributing role in resistance. However, there is little information about the contribution of ST131 to different clinical syndromes or the basis for its impressive emergence and epidemic spread. To investigate this, we studied 953 E. coli isolates from women of reproductive age in the central west region of New South Wales, Australia, including 623 urinary isolates from patients with cystitis (cystitis isolates) (n = 322) or pyelonephritis (pyelonephritis isolates) (n = 301) and 330 fecal isolates from healthy controls. The characteristics studied included ST131 clonal group status, resistance to different antibiotics, presence of virulence factor (VF) genes, and biofilm production. As expected, fecal isolates differed significantly from urinary (cystitis and pyelonephritis) isolates in most of the studied characteristics. Antibiotic resistance was significantly more common in ST131 than in non-ST131 isolates. Both antibiotic resistance and ST131 were more common in pyelonephritis than cystitis isolates and least so among fecal isolates. Within each source group, individual VF genes were more prevalent and VF scores were higher for ST131 than for non-ST131 isolates. For ST131 only, the prevalences of most individual VF genes and VF scores were the lowest in the fecal isolates, higher in the cystitis isolates, and highest in the pyelonephritis isolates. Biofilm production was strongly associated with ST131 status and antibiotic resistance. These results clarify the distribution of the ST131 clonal group and its epidemiological associations in our region and suggest that it exhibits both enhanced virulence and increased antibiotic resistance compared with those of other urinary tract infection (UTI) and fecal E. coli isolates from women of reproductive age.     

Molecular epidemiology of virulence and antimicrobial resistance determinants in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> from hospitalised patients in Kilimanjaro,Tanzania

This study aimed to use whole-genome sequencing to determine virulence and antimicrobial resistance genes in K. pneumoniae isolated from patients in a tertiary care hospital in Kilimanjaro. K. pneumoniae isolates from patients attending Kilimanjaro Christian Medical Centre between August 2013 and August 2015 were fully genome-sequenced and analysed locally. Sequence analysis was done for identification of virulence and AMR genes. Plasmid and multi-locus sequence typing and capsular or capsular (K) typing were performed and phylogeny was done to ascertain K. pneumoniae relatedness. Stata 13 (College Station, TX, 77845, USA) was used to determine Cohen’s kappa coefficient of agreement between the phenotypically tested and sequence-predicted resistance. A total of 16 (47.1%) sequence types (STs) and 10 (29.4%) K types were identified in 30 (88.2%) and 17 (50.0%) of all analysed isolates, respectively. K. pneumoniae ST17 were 6 (17.6%). The commonest determinants were bla_CTX-M-15 in 16 (47.1%) isolates_, bla_SHV in 30 (88.2%), bla_OXA-1 in 8 (23.5%) and bla_TEM-1 in 18 (52.9%) isolates. Resistance genes for aminoglycosides were detected in 21 (61.8%) isolates, fluoroquinolones in 13 (38.2%) and quinolones 34 (100%). Ceftazidime and ceftriaxone showed the strongest agreement between phenotype- and sequence-based resistance results: 93.8%, kappa?=?0.87 and p?=?0.0002. Yersiniabactin determinant was detected in 12 (35.3%) of K. pneumoniae. The proportion of AMR and virulence determinants detected in K. pneumoniae is alarming. WGS-based diagnostic approach has showed promising potentials in clinical microbiology, hospital outbreak source tracing virulence and AMR detection at KCMC.     

Multiclonal epidemic of Klebsiella pneumoniae isolates producing DHA-1 in a Spanish hospital

Between June 2007 and January 2008, 26 Klebsiella pneumoniae isolates carrying bla_DHA-1 on an IncL/M plasmid were obtained from clinical samples at Granollers Hospital, Barcelona, Spain. Three of the isolates also carried a bla_CTX-M-15 gene. The 26 isolates showed 11 pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing showed that PFGE patterns A, B and C belonged to sequence type (ST)17, D to ST13, E to ST427, F and G to ST416, H to ST37, I to ST440, J to ST326, and K to ST428. Results demonstrated the effectiveness of the infection control programme in place at the centre. This study reports the first characterization of STs for bla_DHA-1-producing K. pneumoniae isolates.